JP2006523180A - Novel inhibin-related multi-antigen peptide composition that promotes productivity in birds - Google Patents

Abstract

The present invention provides a novel composition of matter comprising a MAP composition comprising at least two inhibin related peptides linked to a backbone. This composition is immunogenic and promotes productivity when administered to animals, particularly birds, with an acceptable carrier.

Description

  The present invention relates to a novel composition of matter comprising an inhibin-related multi-antigen peptide composition that, together with an acceptable carrier, promotes production capacity when administered to animals, particularly birds.

  Bird species provide a significant source of food, as meat and eggs. Promoting avian productivity is of considerable economic value for industries that currently enjoy high growth due to ever increasing demand for products. Broiler and turkey breeding birds will continue to lay as many hatched eggs as possible to meet consumer demand and remain competitive in meat prices. Therefore, if there is a way to increase spawning even a little, there should be considerable economic benefits.

  As an example, a scenario for one breeding broiler hen to lay 15 more eggs during a single spawning cycle (on the basis of a hen housed) (approximately one year in length) is as follows: It is. In such a case, selling additional birds that have just hatched will result in a surplus of $ 2.00 at the current market chick price ($ 0.16 per chick), and this chick will grow. $ 10 to $ 12.00 should result from further selling chicken obtained from (after taking into account appropriate deductions for feed and fixed costs). The estimated number of breeding broiler hens will exceed 60 million in the United States, so the economic benefit should be approximately $ 750 million +. Meat, including the high income that should be gained by promoting the production capacity of breeding turkeys (and hens including ducks, geese, and quails in the special poultry industry, including the chickens discussed above) The overall economic benefit that can be derived from the meat sector of the poultry industry, including all poultry raised to consume the food, is estimated at $ 1 to $ 2 billion.

  The phrase “promoting productivity” is understood by those skilled in the art to indicate an increase in one or more of the following items in a hen: fertility, ie, early onset of egg production, early onset of maximum egg production, Long-lasting egg laying, increased egg production, or lifetime egg production. The phrase also includes improved feed requirements, improved eggshell quality, or increased tolerance to harmful spawning conditions such as heat shock, overcrowding, malnutrition, and noise. This phrase means an increase in one or more of the following items in males: fertility, ie, spring onset or early onset of sperm production, early onset of maximum sperm production, long duration of sperm production, sperm production Increased quantity (sperm count), increased ejaculation volume, improved sperm survival, increased testosterone production, or increased libido.

  Furthermore, an increase in the production capacity of all poultry is necessary to increase the number of poultry produced for consumption and to improve the efficiency of such production, or the feed demand rate. Thus, there remains a need for compositions or methods that improve or enhance the productivity of all poultry, including chickens, turkeys, ducks, quails, and geese, among others.

  There is also a need for a composition or method that promotes the productivity of migratory birds such as ostriches, emu, and rare birds such as parrots. The parrot is a single eye of a bird that contains a parrot and has antipodes and a strong beak-like beak. A parrot is defined to be a member of any of the bird's parrots (the only family of the parrots) and is distinguished by a short, thick, strong beak-shaped beak.

  The need for a composition or method that promotes productivity is not limited to birds. In many animals, there remains a need for effective compositions or methods that promote productivity. For example, there is a continuing need to promote productivity in most animals bred in the agriculture and livestock industry, such as pigs, cattle, and sheep. There continues to be a need to promote productivity in fur animals such as minks, foxes, otters, ferrets, raccoons, and rodents such as rats, mice, gerbils, and hamsters used for pets and experimental research. There is also a growing need for other animals whose skin is used for decorative purposes.

  Compositions or methods that promote productivity are also needed to increase the number of many animals, such as rare or endangered species, to prevent extinction. Furthermore, there is a continuing need to promote productivity in animals used in races, entertainment, or shows (competitions), such as horses, dogs, cats, zoo animals, circus animals, and the like. There is also a need to promote productivity in humans, as shown by the strong demand for human infertility treatment. Thus, there remains a need for a composition or method that promotes productivity in many animals.

SUMMARY OF THE INVENTION The present invention generally relates to the administration of a composition comprising a multi-antigen peptide (MAP) composition comprising an immunogenic peptide linked to an amino acid backbone together with an acceptable carrier to the animal. The present invention relates to a method for promoting productivity. An effective amount of the MAP composition is administered to the animal so that an immune response against the MAP composition occurs in the animal. It should be understood that the methods of the present invention enhance the productivity of animals that produce inhibin. The peptide is preferably an inhibin related peptide. The animal is preferably a bird. The bird is preferably a poultry. More preferably, the bird is a chicken, turkey, duck, goose or quail. Other preferred birds are migratory birds such as emu, ostrich, rare and cassowary.

  The present invention further comprises a MAP composition comprising at least two inhibin-related immunogenic peptides linked to an amino acid backbone, which when combined with an acceptable carrier, can enhance productivity when administered to animals, particularly birds. It relates to a novel composition comprising. More particularly, the present invention is directed to compositions comprising a MAP composition comprising an immunogenic peptide fragment of the mature α subunit of inhibin protein linked to an amino acid backbone. The MAP compositions of the invention also include one or more mature alpha subunits of inhibin protein linked to the amino acid backbone. The inhibin protein may be from any species, but is preferably an avian inhibin, a mammalian inhibin, a fish inhibin, or a reptile inhibin. The invention also includes inhibin peptides that have been converted so that individual amino acids are conservatively substituted with other amino acids, either natural or non-natural.

  The present invention also promotes productivity in animals by the administration of an MAP composition of the present invention comprising at least two immunogenic peptide fragments of inhibin, or conservative substitutions thereof, linked to a backbone comprising amino acids. Also covers methods. In one embodiment, the method includes administering an effective amount of a MAP composition to the female animal. In other embodiments, the method comprises administering an effective amount of a MAP composition to the male animal. Preferably, the immune response that occurs in the animal is to a MAP composition. More preferably, the immune response generated in the animal is also directed against endogenous inhibin protein produced by the animal, or a fragment thereof.

  The method of the present invention promotes productivity in female animals that produce inhibin, such as mammals, reptiles, fish and birds. Preferably, the method promotes productivity in hens. More preferably, the method promotes productivity in chickens, turkeys, quails, geese, ducks, ostriches, emu, and rares. Surprisingly, the method of the present invention accelerates the onset of spring or spawning in animals. The method of the present invention also surprisingly accelerates the onset of maximum egg production in animals. Furthermore, the method of the present invention increases the egg production of the animal. Furthermore, the method of the present invention surprisingly extends the maximum egg-laying duration in animals. Furthermore, the present invention surprisingly increases the lifetime egg production of animals. The method of the present invention also improves the feed requirement of birds. The method of the present invention is particularly effective for birds of lower weight than their industry standards. Thus, the present invention significantly promotes productivity and, in particular, promotes egg laying of birds that are not optimally managed for feeding. The methods of the present invention also surprisingly reduce or eliminate the impact of such harmful egg-laying conditions on the egg-laying rate of animals exposed to harmful conditions. Such harmful conditions include high temperature, overcrowding, malnutrition, low weight, and noise. The method of the present invention also enhances sexual desire and thus fertility in hens.

  The methods of the present invention also improve productivity in male animals that produce inhibin, such as mammals, reptiles, birds and the like. In preferred embodiments, the methods of the present invention improve productivity in roosters including but not limited to chickens, turkeys, quails, ducks, geese, ostriches, emu, and rares. More particularly, the method of the present invention increases testosterone levels in male animals. Similarly, the method of the invention accelerates the onset of spring or sperm production in male animals. The method of the present invention also accelerates the onset of maximum sperm production in male animals. Furthermore, the method of the present invention surprisingly increases the amount of sperm production (sperm count) by male animals. Furthermore, the method of the invention extends the duration of maximal sperm production in the animal. The method also improves sperm viability in animals. The method of the present invention also improves animal feed requirements. The method of the present invention is particularly effective on animals that are underweight than their industry standards. Thus, the present invention significantly promotes productivity in animals that are not optimally controlled for feeding. Furthermore, the method surprisingly reduces or eliminates the effects of such harmful conditions on sperm production in animals exposed to the harmful conditions. Such harmful conditions include high temperatures, overcrowding, malnutrition, and noise. The method of the present invention also surprisingly enhances the sexual desire of male animals, especially roosters, and thus enhances fertility.

  By promoting the development of male and female animals, especially birds, the present invention also promotes faster harvesting of animals as any product, including its meat that can be consumed. The present invention also reduces the production costs of animals, especially birds, for meat, eggs, or other products by reducing feed requirements.

  Thus, the method of the present invention improves the negative impact on the spawning rate of poultry exposed to harmful spawning conditions. This aspect of the present invention is important because poultry are often raised in open, uncontrolled environments. Poultry is often exposed to harmful conditions, such as underfeeding, high temperatures, and other extreme weather conditions that poultry cannot adapt to, thereby reducing spawning rates in the poultry industry.

  As mentioned above, using the method of the present invention, it is bred in the agricultural and livestock industries such as but not limited to pigs, cattle, sheep, turkeys, quails, ducks, geese, chickens, fish, etc. Most animals; fur animals such as minks, foxes, otters, ferrets, rabbits, raccoons; laboratory animals such as rats, mice, gerbils, guinea pigs; animals whose skins are used for decoration purposes, such as crocodiles and snakes; Unusual or endangered species; horses, dogs, cats, zoo animals, circus animals, etc., animals used for races, entertainment or shows (contests); and any animal that produces inhibin, including humans Promote.

  The methods of the present invention include raptors, parrots, eagles, woodpeckers, owls, sparrows, bursops, cucumbers, squirrels, pigeons, pheasants, damselflys, and herons (herodions) Promote productivity in other birds. More particularly, using the method of the present invention, ostrich, emu, rare, chicken, turkey, duck, goose, quail, partridge, pheasant, kiwi, cassowary, parrot, parakeet, macaw, falcon, eagle, hawk, pigeon It can promote the production ability of bats, parakeets, songbirds, jays, blackbirds, finch, bugs, canaries, toucans, nine bird birds, or sparrows.

  Accordingly, one object of the present invention is to provide a novel MAP composition comprising at least two inhibin related peptides, fragments thereof, or conservative substitutions thereof covalently linked to a backbone comprising amino acids.

  Another object of the present invention is to provide a novel MAP composition comprising the mature α subunit of inhibin, or a conservative substitution thereof, covalently linked to a backbone containing amino acids.

  Another object of the present invention is to provide a novel MAP composition comprising a mature α subunit of inhibin, a fragment thereof, or a conservative substitution thereof, covalently linked to a backbone containing amino acids.

  Another object of the present invention is to provide a novel MAP composition comprising a conservatively substituted inhibin fragment covalently linked to a backbone containing amino acids.

  Another object of the present invention is to provide a novel MAP composition comprising an inhibin peptide fragment covalently linked to a backbone containing amino acids and inducing an immune response in the animal's body when administered to the animal together with an acceptable carrier. It is to be.

  Another object of the present invention is to provide a novel MAP composition comprising a peptide fragment of inhibin covalently linked to a backbone containing amino acids and inducing an immune response in the bird's body when administered to the bird together with an acceptable carrier. It is to be.

  Another object of the present invention is to provide a novel MAP composition comprising a peptide fragment of inhibin covalently linked to a skeleton containing amino acids and inducing an immune response in the hen body when administered to an hen together with an acceptable carrier. It is to be.

  Another object of the present invention is to provide a novel MAP composition comprising a peptide fragment of inhibin covalently linked to a backbone containing amino acids and inducing an immune response in the rooster when administered to the rooster with an acceptable carrier. It is to be.

  One object of the present invention is to provide a novel MAP composition comprising a peptide fragment of inhibin covalently linked to an amino acid-containing backbone that increases production capacity in the bird's body when administered to the bird together with an acceptable carrier. It is to be.

  Another object of the present invention is to provide a novel MAP composition comprising a peptide fragment of inhibin covalently linked to an amino acid-containing backbone, which increases production capacity in the hen when administered to an hen together with an acceptable carrier. It is to be.

  Another object of the present invention is to provide a novel MAP composition comprising a peptide fragment of inhibin covalently linked to an amino acid-containing backbone and increasing production capacity in the rooster body when administered to an rooster with an acceptable carrier. It is to be.

  Another object of the present invention includes a peptide fragment of inhibin covalently linked to a backbone containing amino acids to increase production capacity, wherein the production capacity after administration to a hen with an acceptable carrier. , Promotion of the onset of spring, promotion of onset of egg laying, prolongation of egg laying, increase of egg production in hens, reduction of feed requirement, or time to harvest injected bird chicks as meat Is to provide a novel MAP composition as indicated by the shortening of

  Another object of the present invention is to include a peptide fragment of inhibin covalently linked to the amino acid backbone to increase rooster productivity after administration to roosters with an acceptable carrier, where the potency enhances fertility. I.e., promotion of the onset of spring or sperm production; promotion of maximal sperm production; prolonged duration of sperm production; increased sperm production (sperm count); increased ejaculation; sperm motility and survival Provides a novel MAP composition indicated by increased rate; increased testosterone production; enhanced fertility, ie increased libido, or reduced time to harvest female chicks mated with injected males It is to be.

  An advantage of the present invention is the reduction or elimination of this industry practice of recruiting young males to an old breeding bird population to maintain optimal fertility.

  One object of the present invention is to provide a method for promoting productivity in poultry.

  Another object of the present invention is to provide a method for promoting productivity in chickens.

  Another object of the present invention is to provide a method for promoting productivity in turkeys.

  One object of the present invention is to provide a method for promoting productivity in quail.

  Another object of the present invention is to provide a method for promoting productivity in ducks.

  Another object of the present invention is to provide a method for promoting productivity in geese.

  Another object of the present invention is to provide a method for promoting productivity in reptiles.

  Another object of the present invention is to provide a method for promoting productivity in mammals.

  Another object of the present invention is to provide a method for promoting productivity in fish.

  Another object of the present invention is to provide a method for promoting productivity in humans.

  These and other objects, features, and advantages of the present invention will become apparent after review of the following detailed description of the disclosed embodiments and the appended claims.

DETAILED DESCRIPTION The present invention generally involves administering to an animal a MAP composition in an acceptable carrier consisting of at least two immunogenic fragments of an inhibin protein linked to a backbone comprising amino acids. The present invention relates to a method for promoting productivity. An effective amount of the MAP composition is administered to the animal so that an immune response against the MAP composition occurs in the animal. It should be understood that the methods of the present invention enhance the productivity of animals that produce inhibin. The animal is preferably a bird. More preferably, the bird is a chicken, turkey, quail, goose or duck. Still other preferred birds are pheasants and partridges. Still other preferred birds are migratory birds such as emu, ostrich, rare and cassowary. The present invention further relates to the novel MAP compositions described herein.

  After the following definitions, the composition of the present invention will be described in detail, and then the method of the present invention will be described in detail.

Definitions As used herein, the term “bird” or “poultry” is defined as being a member of the Aves class of animals, characterized as a warm-blooded laying vertebrate that is primarily adapted to fly. . Birds include, but are not limited to, birds of prey, parrots, eagles, woodpeckers, owls, sparrows, pupae, quinacea, scallops, pigeons, pheasants, gangsters, and herons is there. As used herein, the term “birds” is defined as a large group of large, non-flying birds, including several eyes, including Emu, Ostrich, Kiwi, And cassowary. As used herein, the term “parrotidae” is a single eye of a bird that contains a parrot, has antipodes and has a strong beak-like beak. A “parrot” is defined as a member of any of the bird's parrots (the only family of the parrots) and is distinguished by a short, thick, strong beak-shaped beak. As used herein, the term “chicken” refers to chickens used to lay edible eggs such as hens, chickens or broilers raised for meat, and laid for both egg laying and meat. Chick ("chicken raised for dual purposes"). The term “chicken” also refers to a chicken produced by the original breeder, or a chicken parent, its parent, or its parent, laid for laying eggs, edible eggs, or edible egg-laying eggs. It also means a chicken such as a parent.

  As used herein, the term “egg” is defined as female macroscopic cells produced by birds and reptiles, encased in a porous, calcareous or sturdy shell. In the present specification, “production of eggs by birds or reptiles” means behavior of laying eggs of birds or reptiles, or “laying eggs”. The term “egg” is defined as a female gamete, also known as an egg. Thus, egg production in all animals other than birds and reptiles is defined herein as the production of an egg and its release from the ovary, or “ovulation”. Thus, as used herein, the term “egg” is defined as a female large cell encased in a porous, calcareous, or sturdy shell when produced by birds or reptiles; It should be understood that when all other animals produce, they are eggs.

  The terms “onset of egg laying”, “first egg laying” and “spring activation period” for birds are used interchangeably herein and refer to the time when a bird lays its first egg. Thus, as used herein, “promoting initiation” of avian egg laying or spring triggering means inducing the first egg laying earlier than normally occurring in a bird. Similarly, “spring onset” and “onset of sperm production” in males are also used interchangeably. A measurement of the pubic spread (eg, more than 3 fingers) is that the “spring start (first egg) start (day)” is about to occur or may have already occurred It is considered to be a marker of sexual development to foresee. Similarly, T-FIVE is an arbitrary marker or predictor of the spring onset period, indicating that the group reached an average egg production rate of 25% on a given day. Measuring FIRST (first egg-laying age) clearly defines the exact day of the “spring period”.

  The terms “promote productivity”, “improve productivity”, and “increase productivity” are used interchangeably to mean one or more of the following ranges: Yes: Promote fertility; Promote the onset of the spring period (spawning or ovulation in females; sperm production in males); Promote the start of maximum spawning or maximal ovulation in females; Increased egg production in females, or increased sperm production in males; prolonged duration of egg production in females, or prolonged duration of sperm production in males; increased number of lifetime eggs or lifetime ovulation in females; Improve rate; improve eggshell quality; increase ambient temperature, overcrowding, poor or suboptimal nutrition, noise and other harmful conditions; males Improved survival of sperm; increase in testosterone production in males; increased ejaculate volume; enhancement of libido in males and females; animal or its progeny, decrease in the number of age that may be harvested for meat or other animal products.

  The phrase “intensity” is known to those skilled in the art to mean the frequency of egg laying.

  The phrase “lifetime egg production” of a bird is defined as the total number of eggs that the bird lays during its entire lifetime. As used herein, “hen day egg production” or “HDEP” is defined as the average number of eggs a particular hen group lays per day.

  As used herein, the phrase “promoting the start of maximum egg production” or “promoting the start of maximum egg production” means that from hatching, the egg laying or ovulation of the animal is 25% of the maximum egg laying rate or maximum ovulation rate, It means that the time to reach a stage that is less than 50%, 75%, or any other selected maximum or maximum ovulation rate is shorter than the normal period from hatching to maximum laying.

MAP Compositions of the Invention One embodiment of the invention is a multivalent ligand having biological activity that affects productivity and is represented by Structural Formula I.

Wherein B is a multiple linker backbone, n is an integer from 2 to about 20, each L is a covalent bond or linking group, which may or may not be present, and each P is A peptide having about 4 to about 115 amino acid residues. The at least two peptides include a peptide fragment of the inhibin productivity homology region (PPR), a hybrid peptide, a peptide derivative of the hybrid peptide, or a conservative substitution peptide. A PPR is any inhibin peptide region that can elicit an immune response when administered as a peptide component of a MAP composition of the invention. This immune response to PPR, or a fragment or conservative substitution thereof, can increase the productivity of animals that have received a MAP composition comprising PPR, a fragment thereof, or a conservative substitution thereof. Each P and each linker or covalent bond is independently selected and may be the same or different.

Another embodiment of the invention is a polypeptide multivalent ligand having biological activity that affects productivity. A “polypeptide multivalent ligand” is a repeating polypeptide chain in which two or more peptides denoted by P are separated by a multiple linker backbone or peptide spacer denoted by B, respectively. Polypeptide multivalent ligands are represented by structural formulas (II and III).

In the above formula, m is an integer from 0 to about 20.

In the above formula, n is an integer of 1 to 20, preferably 2 to 10, more preferably 3 to 7, and a is 1 or 2. In a preferred embodiment, a is 2 and n is 3. In this embodiment, the two P are linked to each of the first B and the third B in the composition.

The MAP compositions of the present invention may exist in a variety of structures including, but not limited to, linear, cyclic and branched structures. Non-limiting examples of such structures include:

  In the above structural formula of an exemplary embodiment of a MAP composition, each P is an inhibin related peptide having from about 4 to about 115 amino acid residues, y is 1 or 2, and x is an integer from 1 to 3. Yes, n is an integer of 1-20, preferably 2-10, more preferably 3-7. The at least two peptides include peptide fragments or derivatives of inhibin PPR, hybrid peptides, or peptide derivatives of hybrid peptides. Preferably, the peptide comprises mature inhibin α subunit, a peptide fragment or derivative of PPR of mature inhibin α subunit, a hybrid peptide, or a peptide derivative of a hybrid peptide. Such a hybrid peptide or derivative thereof is preferably substantially homologous to the mature inhibin α subunit.

Each B is a skeletal structure consisting of one or more hydrocarbons consisting of at least two amino acids, optionally having from about 2 to about 30 carbons. B preferably consists of at least two amino acids capable of binding to P. Optionally, B may include other amino acids that cannot bind to P, or hydrocarbon chains (CH ₂ ) _n (n is 1 to 20). Such hydrocarbon chains are preferably saturated. Each peptide P and each B are independently selected and may be the same or different.

  When a sufficient number of amino acid residues are identical to each other at corresponding positions in each amino acid sequence, or a peptide having the first amino acid sequence and a peptide having the second amino acid sequence have similar biological activities When structurally related as shown, there is “substantial homology” between the two amino acid sequences in the peptide or protein. The amino acid sequence of the protein has a partial sequence that is substantially homologous to the amino acid sequence of PHI of inhibin, and the partial sequence corresponds to the partial sequence (or the common sequence) when administered to an animal. The protein has a PPR when the peptide having the above is such that the production ability is regulated. In general, when at least 30%, preferably at least 40% of the amino acids in one PPR are identical or structurally related to amino acid residues in the other PPR, the amino acid sequence between the two PPRs There is substantial homology. Structurally such that at corresponding positions in the amino acid sequences (or consensus sequences) of peptides P and PPR, a sufficient number of amino acids are identical to each other or that the peptides can affect productivity. When related, there is substantial homology between the amino acid sequence of peptide P and the amino acid sequence of PPR. In general, at least 40%, preferably at least 50% of the amino acids in the peptide are identical to the amino acid residue at the corresponding position in the PPR, or a subsequence thereof that can affect the production ability or the structure When related above, there is substantial homology between the peptide and PPR. As used herein, “structurally related” is defined below.

  One embodiment of the invention is a peptide derivative of the PPR of any inhibin protein. The peptide derivative has a biological activity that affects productivity when administered to an animal. Examples include peptide derivatives of the peptides represented by SEQ ID NOs: 1-30.

  The “peptide derivative of PPR” includes a peptide fragment of PPR, and the peptide has the amino acid sequence of PPR. The “peptide derivative of PPR” also includes peptides having a sequence corresponding to the PPR fragment. A “PPR fragment” is defined as a peptide whose amino acid sequence corresponds to a partial sequence of PPR, and is called a “partial sequence”. A partial sequence is a contiguous amino acid residue found within a larger sequence.

  In addition, “peptide derivative” refers to one or more amino acid residues in an original sequence or partial sequence, a naturally occurring amino acid residue or amino acid residue analog (also referred to as “modified amino acid residue”) or non-amino acid residue. Peptides having natural amino acid residues and “modified sequences” substituted with amino acid analogs are included. Suitable peptide derivatives are modified sequences that are substantially homologous to the amino acid sequence of PPR or a partial sequence of PPR. Suitable peptide derivatives also include peptides that are substantially homologous to the consensus sequence of the PPR of the inhibin protein.

In one embodiment of the invention, the peptide derivative has an amino acid sequence corresponding to a partial sequence of PPR of about 4 to about 15 amino acid residues. 0, 1, 2, or 3 amino acid residues in the peptide derivative may be different from the amino acid residues in the corresponding positions of the partial sequence of PPR. For example, if the subsequence is

And when one amino acid residue in the sequence of the peptide derivative is different from the amino acid residue in the corresponding position of the subsequence, the peptide derivative is

Wherein [AA '] is a naturally occurring or non-naturally occurring amino acid residue or modified amino acid residue that is different from [AA].

  In another aspect of the invention, the peptide derivative has an amino acid sequence corresponding to the amino acid sequence or partial sequence of PPR of about 4 to about 28 amino acid residues. In other embodiments, the peptide derivative has an amino acid sequence corresponding to the amino acid sequence or subsequence of PPR from about 4 to about 115 amino acid residues. The 0, 1, 2, 3 or 4 amino acid residues in the peptide derivative may be different from the amino acid residues in the corresponding position of the PPR sequence or subsequence.

  An “amino acid residue” is a moiety found in a peptide, represented by —NH—CHR—CO—, where R is the side chain of a naturally occurring amino acid. In this application, the terms “amino acid residue” and “amino acid” are used interchangeably when referring to a moiety found in a peptide. “Amino acid residue analog” includes D-form or L-form having the following formula —NH—CHR—CO—, wherein R is an aliphatic group, substituted aliphatic group, benzyl group, substituted benzyl group, aromatic Is a group or substituted aromatic group and R does not correspond to a side chain of a naturally occurring amino acid.

  Appropriate substitutions of amino acid residues in the sequence of PPR or partial sequence of PPR include conservative substitutions that result in peptide derivatives that can stimulate productivity when administered to animals, preferably birds. A conservative substitution is one in which the substituting amino acid (naturally occurring or modified) is structurally related to the amino acid being replaced, ie, having approximately the same size and chemical properties as the amino acid being replaced. It is a substitution that you have. Thus, the substituting amino acid should have the same or similar functional group in the side chain as the original amino acid.

  “Conservative substitution” also refers to utilizing a substituting amino acid that is identical to the amino acid to be substituted except that the functional group in the side chain is protected with a suitable protecting group. Suitable protecting groups are described in Green and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley and Sons, Chapters 5 and 7, 1991, the teachings of which are incorporated herein by reference. Preferred protecting groups are those that facilitate peptide membrane transport and can be cleaved by hydrolysis or enzymatically, for example, by reducing the hydrophilicity of the peptide and increasing its lipophilicity (Ditter et al., J. Pharm. Sci. 57: 783 (1968), Ditter et al., J. Pharm. Sci. 57: 828 (1968), Ditter et al., J. Pharm. Sci. 58: 557 (1969), King et al., Biochemistry 26 : 2294 (1987), Lindberg et al., Drug Metabolism and Disposition 17: 311 (1989), Tunek et al., Biochem. Pharm. 37: 3867 (1988), Anderson et al., Arch. Biochem. hys. 239: 538 (1985) and Singhal et al., FASEB J. 1: 220 (1987)). Suitable hydroxy protecting groups include ester protecting groups, carbonate protecting groups, and carbamate protecting groups. As described above for the N-terminal protecting group, suitable amine protecting groups include acyl groups and alkoxy or aryloxycarbonyl groups. Suitable carboxylic acid protecting groups include aliphatic, benzyl and aryl esters, as described below for the C-terminal protecting group. In one embodiment, the carboxylic acid group in the side chain of one or more glutamic acid or aspartic acid residues in the peptides of the invention is preferably as a methyl, ethyl, benzyl or substituted benzyl ester, more preferably a benzyl ester. As protected.

  Groups of naturally occurring or modified amino acids that are similar in chemical and steric properties of each amino acid included in each group are provided below. Thus, conservative substitutions can be made by substituting amino acids with other amino acids from the same group. It should be understood that these groups are non-limiting, ie there are further modified amino acids that can be included in each group.

  Group I includes leucine, isoleucine, valine, methionine, and modified amino acids having the following side chains: ethyl, n-propyl, n-butyl. Preferably, Group I includes leucine, isoleucine, valine and methionine.

  Group II includes modified amino acids having side chains of glycine, alanine, valine, and ethyl. Preferably, Group II includes glycine and alanine.

Group III includes phenylalanine, phenylglycine, tyrosine, tryptophan, cyclohexylmethylglycine, and modified amino acids having a substituted benzyl or substituted phenyl side chain. Preferred substituents include one or more of the following: halogen, methyl, ethyl, nitro, —NH ₂ , methoxy, ethoxy and —CN. Preferably, Group III includes phenylalanine, tyrosine, tryptophan.

Group IV includes glutamic acid, aspartic acid, glutamic acid or aspartic acid substituted or unsubstituted aliphatic, aromatic or benzyl esters (eg, methyl, ethyl, n-propyl, isopropyl, cyclohexyl, benzyl, or substituted benzyl) , Glutamine, asparagine, —CO—NH-alkylated glutamine or asparagine (eg, methyl, ethyl, n-propyl and isopropyl), modified amino acids having a side chain of — (CH ₂ ) ₃ —COOH, esters thereof (substituted or Unsubstituted aliphatic, aromatic or benzyl esters), amides thereof, and substituted or unsubstituted N-alkylated amides thereof. Preferably, Group IV includes glutamic acid, aspartic acid, methyl aspartic acid, ethyl aspartic acid, benzyl aspartic acid, methyl glutamic acid, ethyl glutamic acid, benzyl glutamic acid, glutamine, and asparagine.

  Group V includes histidine, lysine, ornithine, arginine, N-nitroarginine, β-cycloarginine, γ-hydroxyarginine, N-amidinocitrulline, 2-amino-4-guanidinobutanoic acid, lysine homolog, arginine homolog, And ornithine homologues. Preferably, Group V includes histidine, lysine, arginine and ornithine. Amino acid homologs include those with 1 to about 3 methylene units added or removed in the side chain.

The Group VI, serine, threonine, cysteine, and straight-chain or branched alkyl side chains of _C 1 -C ₅ substituted with -OH or -SH, for _example, -CH ₂ CH _{2 OH,} - include modified amino acid having a CH ₂ CH ₂ CH ₂ OH or _-CH 2 _CH 2 OHCH _3. Preferably, group VI includes serine, cysteine or threonine.

  In other embodiments, appropriate substitutions of amino acid residues in the sequence of PPR or a subsequence of PPR include “severe” substitutions that result in peptide derivatives that affect productivity. The critical substitutions that result in peptide derivatives affecting productivity are much more likely to be made in highly unconserved positions than in highly conserved positions in the PPR of inhibin proteins.

A “serious substitution” is a substitution in which the substituting amino acid (naturally occurring or modified) has a significantly different size and / or chemical properties than the amino acid to be substituted. Accordingly, the side chain of the amino acid to be substituted may be much larger (or smaller) than the side chain of the amino acid to be substituted and / or has a functional group having a chemical property that is significantly different from the amino acid to be substituted. possible. Examples of this type of critical substitution include substitution of alanine with phenylalanine or cyclohexylmethylglycine, substitution of glycine with isoleucine, substitution of L amino acids with the corresponding D amino acids, or —NH—CH [(— CH of aspartic acid. ₂ ) There is substitution with ₅ -COOH] -CO-. Alternatively, a functional group can be added to the side chain, removed from the side chain, or exchanged with other functional groups. Examples of this type of critical substitution include addition of valine, leucine or isoleucine, exchange of carboxylic acids in the side chain of aspartic acid or glutamic acid with amines, or removal of amine groups in the side chain of lysine or ornithine. There is. In yet another alternative, the side chain of the substituting amino acid may have steric and chemical properties that are significantly different from the functional group of the amino acid being replaced. Examples of such modifications include substitution of glycine with tryptophan, substitution of aspartic acid with lysine, substitution of the side chain of serine with — (CH ₂ ) ₄ COOH. These examples are not necessarily meant to be limiting.

  As used herein, a “hybrid peptide” is an inhibin fragment peptide having about 4 to about 115 amino acid residues. Each amino acid residue (naturally occurring or modified) in the amino acid sequence of the hybrid peptide is (1) identical to the amino acid residue at the corresponding position in the PPR of the original inhibin protein ( 2) an amino acid residue that is structurally related to it, (3) the same amino acid residue at the corresponding position in the PPR of the second inhibin protein, or (4) structurally related to it. It is an amino acid residue. Substitution of an amino acid residue with a structurally related amino acid residue as described above is called “conservative substitution”.

It should be understood that peptide P may be an antigenic peptide derived from any position in the inhibin molecule from any species. In one preferred embodiment, the peptide is derived from the inhibin α subunit, preferably from the mature inhibin α subunit. In another preferred embodiment, the inhibin or inhibin α subunit is an avian inhibin or an avian inhibin α subunit. A preferred embodiment of the present invention is a peptide (P) having an amino acid sequence of AA ₁ to AA ₂₆ represented by SEQ ID NO: 1. This sequence is the 26 amino acids at the N-terminus of the mature chicken inhibin α subunit.

In one embodiment of the invention, four such Ps, each consisting of SEQ ID NO: 1, are linked to the amino acid backbone in a pattern as shown in Formula VII.

In a preferred embodiment, the amino acid backbone is _Bn , where n is 3 and each B is Lys. In this embodiment, two P are linked to Lys ₁ and Lys ₃ , thereby resulting in the structure shown in Structural Formula XIV. This structure is also referred to below as ^1-26 INH-MAP in this application. In this structure, the linking structure (L) is not included, and all Ps are the same and are represented by SEQ ID NO: 1, respectively.

In other preferred embodiments (Formula XV), B-Ala-OH is not present in the central lysine in the amino acid backbone. This structure is also referred to herein as ^1-26 INH-MAP.

  In other embodiments of the present invention, different Ps represented in a structure, for example, structure VII, may be different from each other, and combinations of any number among SEQ ID NOs: 1-30 (below) It's okay. For example, each P in structures I to XIII may be any one of SEQ ID NOs: 1 to 30, or a combination thereof.

  In other embodiments, when P is SEQ ID NO: 1, individual amino acids can be substituted as follows.

AA ₁ is serine, glycine, alanine, cysteine or threonine;
AA ₂ is selected from the group consisting of alanine, threonine, glycine, is a cysteine or serine,
AA ₃ is valine, arginine, leucine, isoleucine, methionine, ornithine, lysine, N- nitro-arginine, beta-cyclo arginine, .gamma.-hydroxy arginine, N- amidino citrulline or 2-amino-4-guanidinobutanoic acid,
AA ₄ is proline, a leucine, valine, isoleucine or methionine,
AA ₅ is, tryptophan, alanine, phenylalanine, tyrosine, or glycine,
AA ₆ is serine, glycine, alanine, cysteine or threonine;
AA ₇ is proline, leucine, valine, isoleucine or methionine;
AA ₈ is alanine, threonine, glycine, cysteine or serine;
AA ₉ is alanine, threonine, glycine, cysteine or serine;
AA ₁₀ is leucine, isoleucine, methionine or valine;
AA ₁₁ is serine, glycine, alanine, cysteine or threonine;
AA ₁₂ is leucine, isoleucine, methionine or valine;
AA ₁₃ is leucine, isoleucine, methionine or valine;
AA ₁₄ is glutamine, glutamic acid, aspartic acid, asparagine, or a substituted or unsubstituted aliphatic or aryl ester of glutamic acid or aspartic acid;
AA ₁₅ is arginine, N-nitroarginine, β-cycloarginine, γ-hydroxyarginine, N-amidinocitrulline or 2-amino-4-guanidinobutanoic acid;
AA ₁₆ is proline, leucine, valine, isoleucine or methionine;
AA ₁₇ is serine, glycine, alanine, cysteine or threonine;
AA ₁₈ is glutamic acid, aspartic acid, asparagine, glutamine, or a substituted or unsubstituted aliphatic or aryl ester of glutamic acid or aspartic acid;
AA ₁₉ is aspartic acid, asparagine, glutamic acid, glutamine, leucine, valine, isoleucine, methionine, or substituted or unsubstituted aliphatic or aryl esters of glutamic acid or aspartic acid,
AA ₂₀ is valine, arginine, leucine, isoleucine, methionine, ornithine, lysine, N-nitroarginine, β-cycloarginine, γ-hydroxyarginine, N-amidinocitrulline or 2-amino-4-guanidinobutanoic acid;
AA ₂₁ is alanine, threonine, glycine, cysteine or serine;
AA ₂₂ is alanine, threonine, glycine, cysteine or serine;
AA ₂₃ is histidine, serine, threonine, cysteine, lysine or ornithine;
AA ₂₄ is a threonine, aspartic acid, serine, glutamic acid, or a substituted or unsubstituted aliphatic or aryl ester of glutamic acid or aspartic acid,
AA ₂₅ is asparagine, aspartic acid, glutamic acid, glutamine, leucine, valine, isoleucine, methionine, or substituted or unsubstituted aliphatic or aryl esters of glutamic acid or aspartic acid,
AA ₂₆ is cysteine, histidine, serine, threonine, lysine or ornithine.

  The 26 examples of amino acid substitutions are not limited to SEQ ID NO: 1, and any of these amino acids may be used as long as the MAP composition of the present invention is effective in promoting productivity when administered to an animal. It should be understood that these examples can be applied to any inhibin fragment, preferably its α subunit, or its α subunit fragment using P in its MAP composition whenever P is present.

  Other peptides that can be used as P in the MAP composition of the present invention include, but are not limited to, the following SEQ ID NOs: 1-30. It should be understood that the P in a given MAP composition can be the same or different.

In other embodiments, the four Ps in structure VII, or two or more Ps in any structure (formulas I-XIII) can be the same or different and can be selected from: .

In other embodiments, the four Ps in structure VII, or two or more Ps in any structure (formulas I-XIII) can be the same or different and can be selected from: .

In other embodiments, the four Ps in structure VII, or two or more Ps in any structure (formulas I-XIII) can be the same or different and can be selected from: .

  In other embodiments, each amino acid residue of the hybrid peptide is the same as the amino acid residue at the corresponding position of the inhibin PPR or fragment thereof, or is a conservative substitution of this residue. Hybrid peptides are included.

Preferably, the amino acid sequence of the hybrid peptide includes a partial sequence derived from the first PPR and a partial sequence derived from the second PPR. Each partial sequence has about 4 to about 115 amino acid residues, preferably about 4 to about 75 amino acid residues, and more preferably about 4 to about 30 amino acid residues. The two partial sequences may be equal in length, for example, both partial sequences may be 9-mer or 12-mer. Or these two partial arrangement | sequences may differ in length, for example, 12-mer and 13-mer may be sufficient. An example is a hybrid peptide represented by SEQ ID NO: 29, which is a 22-mer, in which the first 11 amino acids correspond to the partial sequence of SEQ ID NO: 1, and the second 11 Amino acids correspond to different subsequences of SEQ ID NO: 1.

Another example is a peptide represented by SEQ ID NO: 30, which is a 22-mer, in which the first 11 amino acids correspond to different subsequences of SEQ ID NO: 1, 11 amino acids correspond to the other partial sequence of SEQ ID NO: 1.

  Another embodiment of the present invention is a peptide derivative of a hybrid peptide, such as a peptide derivative of the peptide represented by SEQ ID NO: 29 or SEQ ID NO: 30. In general, fragments of hybrid peptides having at least about 10 amino acid residues and capable of affecting production performance are included in the definition of “peptide derivatives of hybrid peptides”. A “peptide derivative of a hybrid peptide” also includes a peptide having a “modified sequence” in which one or more amino acids in the hybrid peptide are replaced with naturally occurring amino acids or amino acid analogs (also referred to as “modified amino acids”). included. Suitable modified sequences are those that are substantially homologous to the amino acid sequence of the hybrid peptide or a partial sequence thereof, which can affect production performance. Generally, peptide derivatives are about 10 to about 30 amino acid residues.

  In one aspect of the invention, the peptide derivative of the hybrid peptide, eg, SEQ ID NO: 29 or SEQ ID NO: 30, corresponds to a partial sequence of the hybrid peptide of about 10 to about 15 amino acid residues that can affect productivity. It has an amino acid sequence. The 0, 1, 2, or 3 amino acid residues in the peptide derivative may be different from the amino acid residues at corresponding positions in the partial sequence of the hybrid peptide.

  In another aspect of the invention, the peptide derivative has a productivity of about 4 to about 115 amino acid residues, preferably about 4 to about 75 amino acid residues, more preferably about 4 to about 30 amino acid residues. It has an amino acid sequence corresponding to the amino acid sequence of the hybrid peptide or a partial sequence thereof, which can influence. The 0, 1, 2, 3 or 4 amino acid residues in the peptide derivative may be different from the amino acid residues at corresponding positions in the sequence or partial sequence of the hybrid peptide.

In the present specification, the aliphatic group, straight chain, branched chain, or contain hydrocarbon cyclic C ₃ -C _9, which is fully saturated, nitrogen, oxygen, heteroaryl such as sulfur It contains one or two atoms and / or contains one or more unsaturated bonds. Aromatic groups include aromatic carboxylic acid groups such as phenyl and naphthyl, and heterocyclic aromatic groups such as imidazole, indolyl, thienyl, furanyl, pyridyl, pyranyl, oxazolyl, benzothienyl, benzofuranyl, quinolinyl, isoquinolinyl, acridinyl and the like. included.

Suitable substituents on aliphatic, aromatic or benzyl groups include, for example, —OH, halogen (—Br, —Cl, —I, and —F), —O (aliphatic group, substituted aliphatic Group, benzyl group, substituted benzyl group, aryl group or substituted aryl group), —CN, —NO ₂ , —COOH, —NH ₂ , —NH (aliphatic group, substituted aliphatic group, benzyl group, substituted benzyl group) , Aryl group or substituted aryl group), —N (aliphatic group, substituted aliphatic group, benzyl group, substituted benzyl group, aryl group or substituted aryl group) ₂ , —COO (aliphatic group, substituted aliphatic group, benzyl) group, substituted benzyl group, an aryl group or a substituted aryl _group), - CONH _2, -CONH (aliphatic group, substituted aliphatic group, benzyl group, substituted benzyl group, an aryl group or a substituted aryl group), - SH, -S Aliphatic group, substituted aliphatic group, benzyl group, substituted benzyl group, there are aromatic or substituted aromatic group) and -NH-C (= NH) -NH 2. A substituted benzyl or aromatic group can also have an aliphatic group or a substituted aliphatic group as a substituent. The substituted aliphatic group can also have a benzyl group, a substituted benzyl group, an aryl group or a substituted aryl group as a substituent. The substituted aliphatic group, substituted aromatic group or substituted benzyl group can have a plurality of substituents.

  A “multivalent ligand” is a molecule having a series of peptides represented as “P” in the structural formula shown above and throughout the application. In this application, the term multivalent ligand is considered equivalent to the term multiple antigen peptide (MAP) composition. Preferably, the multivalent ligand or MAP comprises 2 to about 30 peptides. Each peptide is linked to the multilinker backbone by a covalent bond or by a linking group. Each peptide derivative and each linker or each covalent bond is independently selected. Non-limiting examples of multivalent ligands are represented by structural formulas I-XV.

  A “polypeptide multivalent ligand” is a plurality of repeating polypeptide chains in which two or more peptides P are each separated by a peptide linking group. Polypeptide multivalent ligands are represented by structural formulas II and III.

  The at least two peptides represented as P in the multivalent ligand or polypeptide multivalent ligand of the present invention may be a hybrid peptide, a peptide derivative of a hybrid peptide, a peptide derivative of PPR, or a combination thereof. A multivalent ligand or polypeptide multivalent ligand can also have one or more other peptides that do not substantially reduce the ability to affect production capacity. However, it is preferred that all peptides in the multivalent ligand or polypeptide multivalent ligand are hybrid peptides, peptide derivatives of hybrid peptides, and / or peptide derivatives of PPR. Optionally, the C-terminus or N-terminus of the polypeptide multivalent ligand can include an amino acid sequence that can aid in its separation or isolation, such as a hemagglutinin antigen, or polyhistidine sequence. A multivalent ligand or polypeptide multivalent ligand can have peptides that are derivatives of different PPR and / or peptides that are derivatives of the same PPR but have different amino acid sequences. A multivalent ligand or polypeptide multivalent ligand can have a peptide that is a hybrid consisting of a set of different PPRs and / or a peptide that is a hybrid of two identical PPRs but has a different amino acid sequence. Similarly, a multivalent ligand or polypeptide multivalent ligand can have peptides that are derivatives of different hybrid peptides and / or peptides that are derivatives of the same hybrid peptide but have different amino acid sequences. The peptides of the multivalent ligand or polypeptide multivalent ligand may all be the same.

Multiple Linker Skeleton The multiple linker skeleton represented as “B” in the structures included herein is a linear or branched molecule having a number of appropriately spaced reactive groups, each of which The group can react with a functional group in the peptide or linker. Suitable multi-linker scaffolds are biocompatible and suitable for administration such as parenteral or oral administration after attachment of the peptide derivative. In general, the multilinker backbone has a molecular weight of less than about 20,000 atomic mass units (amu) and usually contains from 2 to about 100 attachment sites. Not all attachment sites need to be occupied.

  Reactive functional groups in the multiple linker backbone are used as attachment sites for peptides or linkers. Attachment sites are “suitably spaced” when the covalent bond formation between certain reactive functional groups and the peptide is not substantially inhibited by steric hindrance.

  Suitable reactive groups in the multilinker backbone include amines, carboxylic acids, alcohols, aldehydes, thiols. The amine group in the multilinker backbone can form a covalent bond with the C-terminus of the peptide derivative or with the carboxylic acid functional group in the linking group. Carboxylic acid groups or aldehydes in the multilinker backbone can form a covalent bond with the N-terminus of the peptide derivative or an amine group in the linking group. The alcohol group in the multilinker backbone can form a covalent bond with the C-terminus of the peptide derivative or with the carboxylic acid functional group in the linking group. A thiol group in the multilinker backbone can form a disulfide bond with a cysteine in a peptide derivative or a thiol group in a linking group. As described above, a bond can also be formed between a reactive functional group in the multiple linker backbone and an appropriate functional group in the amino acid side chain of the attached peptide. The functional group that links each peptide to the multiple linker backbone may be different for all peptides, but is preferably the same.

  Examples of suitable polypeptide multivalent scaffolds are disclosed in Tam, Journal of Immunological Methods 196: 17 (1996), the entire teachings of which are incorporated herein by reference. In general, a suitable polypeptide multilinker backbone is from about 1 to about 20 amino acid residues, preferably 2 to 10 amino acid residues, more preferably 3 to 7 amino acid residues. As with other multilinker backbones, it usually has from about 2 to about 20 attachment sites, which are often functional groups located in the side chains of amino acid residues. However, alpha amino groups and alpha carboxylic acids are also used as attachment sites.

  Preferred polypeptide multilinker backbones represented by “B” in the structure shown above include polylysine, polyornithine, polycysteine, polyglutamic acid and polyaspartic acid, or mixtures thereof. Optionally, amino acid residues having an inert side chain, such as glycine, alanine, and valine, may also be included in the amino acid sequence. The polypeptide may be pennant or cascading. A “pennant polypeptide” is linear. As is the case with polypeptides normally found in nature, the amide bond of a pennant polypeptide is formed between the α amine of one amino acid residue and the α carboxylic acid of the next amino acid residue. When n is less than 5, there are usually 0-6 amino acids between the attachment sites, and when n is greater than 5, there are usually 1-6 amino acids between the attachment sites. A “cascade polypeptide” is branched and contains at least some of the amino acid groups between the side chain functional group of one amino acid residue and the α amino group or α carboxylic acid group of the next amino acid residue. An amide bond is formed. For example, at least some amide bonds of the cascade polylysine are formed between the ε-amine group of the lysine residue and the carboxylic acid residue of the next lysine residue.

In one embodiment, the backbone “B” consists of 3 amino acids, which may be Lys, Asp, Glu, Cys, Orn, Gly, Ala and Val, or a mixture thereof. Thus, embodiments of B include, but are not limited to:

Linker (L)
A suitable linker (L) is a group that can link a peptide derivative to a multiple linker backbone. In one embodiment, the linker is an oligopeptide of about 1 to about 10 amino acids and consists of amino acids having an inert side chain. Suitable oligopeptides include polyglycine, polyserine, polyproline, polyalanine, and oligopeptides consisting of alanine and / or serine and / or proline and / or glycine amino acid residues. In other embodiments, the _linker, X 1 - is a polyethylene glycol _{-X 2 - (CH 2) m} -X 2 or _{X 1.} X ₁ and X ₂ are functional group residues, each covalently linked to an appropriate functional group residue in the multilinker backbone or peptide derivative. Examples of X ₁ and X ₂ are: 1) the residue of an alcohol group that forms an ester with the residue of a carboxylic acid group in the multiple linker backbone or peptide derivative; 2) the carboxyl in the multiple linker backbone or peptide derivative Residue of amine group that forms amide with acid group residue, 3) Residue of carboxylic acid or aldehyde group that forms amide with amine residue in multiple linker skeleton or peptide derivative, or 4) Multiple linker There are residues of thiol groups that form disulfides with residues of thiol groups in the backbone or peptide derivatives. m is an integer from 2 to about 20.

  The peptides in the multivalent ligand can be attached to the multiple linker backbone by covalent bonds, linker groups, or combinations thereof. The linker groups may be the same or different. Preferably, any peptide in the multivalent ligand is linked to the multilinker backbone by covalent bonds. Alternatively, every peptide in the multivalent ligand is linked to the multiple linker backbone by the same linking group, eg, a glycine residue, or a glycine-glycine dipeptide.

  The multivalent ligand includes a 4-branched pennant-type polylysine trimer, and four peptides may be attached to a linear trimer polypeptide skeleton composed of 3 lysine residues (Lys Lys Lys). It is suggested. This attachment is performed using a peptide bond between the C-terminus of each peptide or linking oligopeptide and the amino group in one of the three lysine side chains or the N-terminus of polylysine. All peptides and all linkers in a given multivalent ligand may be the same or different.

The polypeptide spacer in the multilinker backbone B shown in Structural Formula (II) is a peptide having from about 5 to about 40 amino acid residues. The spacers in the polypeptide multivalent ligand are each independently selected, but are preferably all the same. The spacer should be able to move flexibly within the space of the adjacent peptide P and is therefore usually rich in small amino acids such as glycine, serine, proline or alanine. The peptide spacer preferably comprises glycine or alanine, preferably at least 60%, more preferably at least 80%. Furthermore, peptide spacers generally have little or no biological and antigenic activity. Preferred spacers are (Gly-Pro-Gly-Gly) _x , and (Gly ₄ -Ser) _y , where x is an integer from about 3 to about 9, and y is from about 1 to about 8 Is an integer. A specific example of a suitable spacer is (Gly ₄ -Ser) ₃ .

The spacer can also include 1 to about 4 amino acids that create a proteolytic cleavage signal.

  The multivalent ligand or polypeptide multivalent ligand of the invention can be administered concurrently with other pharmaceutically active agents. In one example, the multivalent ligand or polypeptide multivalent ligand is administered concurrently with other drugs normally administered to birds.

  The peptide derivatives, multivalent ligands and / or polypeptide multivalent ligands of the present invention have many uses besides increasing productivity. Some of this usage is discussed in the following paragraphs.

  The disclosed multivalent ligand can be used to produce both polyclonal and monoclonal antibodies directed against peptide derivatives attached thereto. Methods for producing antibodies against peptide antigens attached to multiple linker scaffolds are described in Tam, Proc. Natl. Acad. Sci. USA 85: 5409 (1988), Tam and Lu, Proc. Natl. Acad. Sci. USA 86: 9084 (1989) and Tam, Journal of Immunological Methods 196: 17 (1996).

  Appropriate antibodies can also be produced by optionally combining the peptide derivatives and hybrid peptides of the present invention with an appropriate carrier such as mussel hemocyanin or serum albumin. Polyclonal and monoclonal antibody production can be performed using any suitable technique. Some of the peptides, peptide derivatives and hybrid peptides of the present invention are sufficiently antigenic and do not require a carrier for antibody production. Various methods of monoclonal antibody production have been described (eg, Kohler et al., Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6: 511-519 (1976); Milstein et al., Nature 266: Korowski et al., U.S. Patent No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory: Cold Spring N. Cold Spring. ); Current Protocols in Molecular Biology, Vol. 2 (Supplement 27, Summer 1994) Ausubel, FM, et al., Eds. (See John Wiley & Sons: New York, NY), Chapter 11, (1991)). In general, hybridomas can be produced by fusing a suitable immortal cell line (eg, a myeloma cell line such as SP2 / 0) with antibody producing cells. Antibody producing cells, preferably spleen or lymph node producing cells can be obtained from animals immunized with the antigen of interest. The cells (hybridomas) can be isolated using selective culture conditions and can be cloned by limiting dilution. Cells that produce antibodies with the desired specificity can be selected by an appropriate assay (eg, ELISA).

  There are a variety of uses for antibodies to PPR of inhibin protein and fragments thereof, including monoclonal antibodies. For example, determining whether inhibin protein is present in a liquid sample obtained from a test animal using an antibody directed against or reactive to the inhibin protein, preferably an antibody that specifically binds to the PPR of said protein. Can do. The sample is treated with an anti-PPR antibody specific for the inhibin protein or fragment thereof. The sample is then analyzed for complex formation between the inhibin protein and the antibody, for example, by Western blotting or immunoprecipitation. The sample may be, for example, a clear lysate of cells, which is treated with a detergent such as sodium deoxycholate (0.5% to 1%) or sodium dodecyl sulfate (1%). Obtained by centrifuging and separating the supernatant from the pellet.

  As demonstrated by the fact that multivalent ligands, including peptide derivatives or hybrid peptides of the present invention, have such dramatic effects on biological processes such as productivity, PPR is the biological of inhibin protein. Plays an important role in activity. The polypeptides multivalent ligands, peptide derivatives and hybrid peptides of the present invention can also be used to identify molecules that interact with the PPR of a particular inhibin protein and whose activity is modulated by this PPR. For example, an affinity column can be prepared in which a specific polypeptide multivalent ligand, peptide derivative or hybrid peptide is covalently linked directly or via a linker. This column can then be used to isolate and identify specific molecules that bind to the PPR of the inhibin protein and are likely to also bind to the inhibin protein from which the peptide derivative or hybrid peptide is derived. The molecule can then be eluted from the column, characterized and tested for the ability to interact with the inhibin protein or fragment thereof. Such peptides may be useful as the peptide component of the MAP composition taught in the present invention.

Synthesis of Peptide Sequences and MAPs Peptide sequences in the compounds of the present invention are known to those skilled in the art, including solid phase peptide synthesis (eg BOC or FMOC) methods, liquid phase synthesis methods, or combinations of the above methods It can be synthesized by other suitable techniques. Established and widely used BOC or FMOC methods are described in Merrifield, J. et al. Am. Chem. Soc. 88: 2149 (1963); Meienhofer, Hormonal Proteins and Peptides, C.I. H. Li, Ed. , Academic Press, 1983, pp. 48-267; and Barany and Merrifield, in The Peptides, E .; Gross and J.M. Meienhofer, Eds. Academic Press, New York, 1980, pp. 3-285. Solid phase peptide synthesis is described in Merrifield, R .; B. Science, 232: 341 (1986); Carpino, L .; A. And Han, G .; Y. J. et al. Org. Chem. 37: 3404 (1972); and Gauspohl, H .; Et al., Synthesis, 5: 315 (1992).

  Multivalent ligands are described in Tam, J. et al. , Immunological Methods 196: 17 (1996), Kim et al., Cancer Research 54: 5005 (1994), Nomizu et al., Cancer Research 53: 3459 (1993) and Tam, J. et al. Proc. Natl. Acad. Sci. USA 85: 5409 (1989), and can be prepared by methods known to those skilled in the art.

  A convenient way to prepare multivalent ligands in a single operation is to reach the desired branched chain, for example, bifunctional protected Boc-Lys (Boc) in Boc chemical synthesis, or two in Fmoc chemical synthesis. This is by stepwise solid phase synthesis starting from a C-terminal core matrix using a functional group protected Fmoc-Lys (Fmoc). The selected peptide derivative, hybrid peptide or hybrid peptide derivative is then sequentially extended into a lysine core matrix on the resin to form the desired multivalent ligand. This stepwise method generates multivalent ligands in the C → N orientation. Chimeric multivalent ligands to which two or more different peptides have been added can also be produced in this way by synthesizing both sequences in tandem. Alternatively, different peptides can be synthesized on different arms of the core matrix using a core matrix containing two different amine protecting groups. Methods have been developed to distinguish lysine α- and ε-amines so that different peptides and functional moieties can be introduced. The general theme in these methods is to manipulate the orthogonality or the differential tendency of the deprotection method. Suitable combinations include (i) Boc-Fmoc (Tam and Lu, Proc. Natl. Acad. Sci., USA 25 86: 9084 (1989)), (ii) Fmoc-Dde (Bycroft et al., J. Chem. Soc.Chem.Commun. 1993: 773 (1993), and (iii) Npys-Fmoc (Ahlburg, J. Immunol. Methods 179: 269 (1995)) Other approaches to preparing polypeptide multivalent ligands. Rotzschlee et al., Proc. Natl. Acad. Sci. USA 94: 1462 (1997).

  Recombinant and purification techniques known to those skilled in the art using expression systems to make peptides and proteins can be used to make and subsequently purify the peptides used in the MAP compositions of the invention. it can.

Preparation of MAP protein A 4-branched multilinker skeleton (B) to which a PPR peptide was added was synthesized by t-butoxycarbonyl (Boc) βAla-OCH ₂ — in which 0.05 mmol of βAla was present in 0.5 g of resin. Stepwise solid phase procedure (Merifield, RB, J. Am. Chem. Soc) on Pam resin (Mitchell, AR, et al., J. Org. Chem. 43: 2845-2852 (1978)). 85: 2149-2154 (1963)). All initial and subsequent syntheses at the carrier core level are pre-formed symmetry of N ^α , N ^ε -Boc-Lys (Boc) in dimethylformamide (12 ml HCONMe ₂ per gram of resin) greater than 4M. Anhydrous anhydrides (sequentially 0.2, 0.4, 0.8 and 1.6 mmol) followed by a second coupling with only dicyclohexylcarbodiimide in CH ₂ Cl _2. Thus, after deprotection, a 4-branched core matrix containing 4 functional amino groups was obtained. Protecting groups for peptide antigen synthesis are Boc groups for the α-amino terminus and benzyl alcohol derivatives for most side chain amino acids. For all residues except arginine, asparagine, glutamine, and glycine, CH ₂ Cl while monitoring with a quantitative ninhydrin test (Sarin, VK, et al., Anal. Biochem., 117: 147-157 (1981)). ₂ with a pre-formed symmetrical anhydride for 1 hour and a _second coupling in HCONMe ₂ (if necessary) at 50 ° C. with N A third coupling was performed in methylpyrrolidone (Tam, JP, in Proceedings of the Ninth American Peptide Symposium, eds. Deber, CM, Kople, KD and Hruby, V. J. (Pierce Chem., Rockford, III pp.305-308 (1985)). The coupling of Boc-Asn and Boc-Gln was mediated by preformed 1-hydroxybenzotriazole ester in HCONMe ₂ . Boc-Gly and Boc-Arg were coupled using only water-soluble dicyclohexylcarbodiimide to avoid the risk of forming dipeptides and lactams, respectively. Acetylation in 3 mM acetic anhydride in HCONMe ₂ containing 0.3 mmol of N, N-dimethylaminopyridine upon completion of the multivalent ligand to remove polycationic amino groups that yield highly charged macromolecules As a result, a cap was formed on the α-amino group of the peptide chain. The deprotection process was initiated by removing the dinitrophenyl protecting group of His (Dnp) with 1M thiophenol in HCONMe ₂ over 8 hours (3 times at 50 ° C. if necessary to complete the reaction). . Cleavage to yield unpurified multivalent ligand (cleavage yield 85% to 93%) low-high-HF method or low-high-trifluoromethane-sulfonic acid method (Tam, JP et al., J. MoI. Am. Chem. Soc., 108: 5242-5251 (1986)), the branched peptide oligolysine was cleaved from the crosslinked polystyrene resin support. The crude peptide and resin were then washed with cold ether / mercaptoethanol (99: 1, volume / volume, 30 ml) to remove p-thiocresol and p-cresol, and 8M urea / 0.2M dithiothrei Peptides were extracted using 100 ml of Toll / 0.1 M Tris-HCl buffer, pH 8.0. In order to remove any remaining aromatic by-products generated in the cleavage step, 8 M urea, 0.1 M NH ₄ HCO ₃ (NH ₄ ) ₂ CO ₃ buffer in a Spectrum Por 6 tube with a cutoff molecular weight of 1000 MW. The peptide was dialyzed by equilibrating at 0 ° C. for 24 hours with a degassed, N ₂ purged solution containing solution, pH 8.0, 0.1 M mercaptoethanol. The dialysis was then continued for 12 hours in 8M urea, then in 2M urea, all in pH 8.0, 0.1M NH ₄ HCO ₃ / (NH ₄ ) ₂ CO ₃ buffer, Dialysis was performed sequentially in H ₂ O and 1M HOAc to remove all. The multivalent ligand was lyophilized and purified batchwise by high performance gel permeation chromatography or high performance ion exchange chromatography. All purified material was analyzed and found to contain the predicted amino acid sequence.

  Another important feature of the multilinker backbone (B) as an antigen carrier is known for its exact structure; it is itself antigenic and may cause tissue irritation or other undesirable reactions There is no impurity; the exact concentration of inhibin peptide (s) (P) is known; P is symmetrically or asymmetrically distributed on the backbone; and can produce multivalent vaccines It can be used as a base for such multiple antigens. One of the major advantages of the MAP composition of the present invention as the basis for an inhibin vaccine is that it differs from previous systems using natural carriers such as mussel hemocyanin, maltose binding protein, tetanus toxoid, and bovine serum albumin, The multiple linker backbone (B) of the present invention is a fully defined chemical moiety that binds inhibin peptide antigens at known concentrations. Furthermore, this inhibin peptide antigen occupies a large portion of the molecule, not a relatively small undefined portion of the molecule, as is the case with large natural carriers such as maltose binding proteins.

  When MAP molecules are used for vaccine production, the multiple linker backbone is preferably a naturally occurring amino acid such as lysine so that it can be processed in the body following normal metabolic pathways. However, as explained more fully below, non-naturally occurring amino acids, and even non-alpha amino acids, can be used. Amino acids or other asymmetric molecules used in constructing the skeleton may be either D-form or L-form.

Although the dendritic polymer has been primarily described above as a polyamide polymer, it will be readily apparent that the multiple linker backbone (B) of the present invention is not limited to dendritic polyamides. Any of a wide variety of molecules having at least two available functional groups can be used as the multilinker backbone. For example, propylene glycol can be used as the basis for a polyester dendritic polymer. Succinic acid with a selected glycol or amine can be used as a core molecule to produce a polyester or polyamide. Diisocyanates can be used to produce polyurethanes. The important point is that the multilinker backbone has at least two available functional groups, from which there are also at least two available functional groups or anchoring groups on each branch. This means that the same branched chain can be produced by a continuous scaffold-forming type reaction with the additional molecules it has. Immobilization that can immobilize the peptide (P) or linker (L) in the simple case where the multiple linker backbone has two available functional groups and each subsequent generation also has two available functional groups The number of parts is represented by (2) ⁿ (n is the number of generations).

  For a more complete discussion of dendritic polymer chemistry, see Tomalia et al., Polymer Journal 17 (1), 117 (1985), Aharoni et al., Macromolecules 15, 1093 (1982), and U.S. Pat. No. 4,289,872. No. 4,376,861, No. 4,507,466, No. 4,515,920, No. 4,517,122, No. 4,558,120, No. 4,568,737, Note Nos. 4,587,329, 4,599,400, and 4,600,535.

  In a presently preferred embodiment, the present invention provides a multilinker backbone (B) having multiple anchoring sites covalently linked to the same or different peptide (P) and optionally a linker between the peptide and the backbone (L) and a plurality of antigen peptide systems. The multilinker backbone has at least two functional groups to which molecular branches with terminal functional groups are covalently bonded. The terminal functional group on the branched chain is covalently bonded to the peptide (P). In the present specification, the antigen molecule P has been mainly described as a peptide antigen, but this is not limited to a peptide antigen, and even not to an antigen.

  The selected peptide (P) can be separately synthesized or obtained by other methods and combined with the backbone (B). Alternatively, P can be synthesized on the skeleton. For example, if P is an oligopeptide or a relatively low molecular weight polypeptide and the available functional group on B is an amino group or a carboxyl group, each branched chain of B can be defined using known peptide synthesis techniques. By stretching, P can be synthesized.

  A particular advantage of the present invention is that B can be used as a support for two or more different Ps. P may be all related to inhibin or may be a mixture of an inhibin related peptide and other peptides. This is particularly useful for the generation of MAP vaccines, or for the generation of MAP vaccines that contain one or more Ps and one or more antigens useful for protecting against avian diseases. Vaccines generated from antigen products of the present invention in which T cell antigens and B cell antigens related to the same disease are attached to the backbone polymer in any of various structures are useful because they can generate high antibody titers. It is.

  One embodiment of the present invention that utilizes this approach is on polylysine or other structures that use different amino blocking groups, one being stable to acid hydrolysis and the other being stable to alkali hydrolysis. Based on using a similar molecule as B. This makes it possible to protect either of the amino groups of lysine by an orthogonal protection method.

  Fluorenylmethyloxycarbonyl (Fmoc) is a base labile protecting group and is completely stable to acid deprotection. The t-butoxycarbonyl blocking group (Boc) is stable under basic conditions but not stable under weakly acidic conditions such as 50% trifluoroacetic acid. One set on the α-amino group of lysine by selecting Boc-lys (Boc) -OH, Boc-lys (Fmoc) -OH, Fmoc-lys (Boc) -OH or Fmoc-lys (Fmoc) -OH Other antigens can be placed on the ω-amino group. One skilled in the art of peptide synthesis can easily devise methods to achieve the same type of product using a variety of blocking groups and other dendritic polymers.

The following abbreviations are used in the examples.
Boc-13-t-butoxycarbonyl TFA-trifluoroacetic acid DMF-dimethylformamide DCC-dicyclohexylcarbodiimide Tos-tosyl Bzl-benzyl Dnp-dinitrophenyl 2CIZ-2-chlorocarbobenzoxy DIEA-diisopropylethylamine TFMSA-trifluoromethylsulfonic acid BSA-Bovine serum albumin HPLC-High performance liquid chromatography TBR-Tumor bearing rabbit ATP-Adenosine triphosphate Dnp-Dinitrophenyl CIZ-Chlorobenzyloxycarbonyl BrZ-Bromobenzyloxycarbonyl ELISA-Enzyme-linked immunosorbent assay

Surprisingly, it has been discovered that the composition of the present invention promotes animal productivity, particularly avian productivity. Accordingly, the present invention is also directed to a method of promoting production performance in animals by administering a MAP composition of the present invention with an acceptable carrier. In one embodiment, the method comprises administering to a female animal an effective amount of a MAP composition with an acceptable carrier so that the animal's production capacity is increased. In other embodiments, the method includes administering to the male animal an effective amount of the MAP composition with an acceptable carrier so that the animal's production capacity is increased. Preferably, an immune response against the MAP composition occurs in the animal body. More preferably, the immune response generated in the animal is also directed to the inhibin protein (endogenous inhibin) produced by the animal.

  More specifically, the methods of the present invention comprise administering to an animal an effective amount of a MAP composition of the present invention so that the animal's productivity is enhanced. Preferably, the animal is a bird. A preferred bird is poultry. Particularly preferred birds are chickens, quails, turkeys, geese and ducks. It should be understood that a “treated” bird is a bird that has been administered a MAP composition of the invention.

  The method of the present invention can be used to promote the productivity of any species of hen that produces inhibin. The hens include, but are not limited to, birds of prey, parrots, eagle hawks, woodpeckers, owls, sparrows, bursops, quininas, staghorns, pigeons, pheasants (poultry), geese (geese) , Ducks, other waterbirds), and herons. More specifically, hens include, but are not limited to, ostrich, emu, rare, kiwi, cassowary, turkey, quail, chicken, falcon, eagle, hawk, pigeon, parakeet, butterfly, macaw, parrot, passerine Are birds (songs, jays, blackbirds, finch, bugs, sparrows, etc.), and any bird belonging to the order of the parrot. A preferred bird is poultry. Particularly preferred poultry are chickens, quails, turkeys, ducks or geese. The method of the present invention can also be used to promote spawning initiation in endangered birds. Such endangered birds include, but are not limited to, grouse, giant grouse, eagle, hawk, condor, and owl.

  The peptide fragment (P) of inhibin in the MAP composition of the present invention may be derived from inhibin from any animal species that produces inhibin. The peptide fragment (P) in a given MAP composition may be derived from various species. Inhibins include, but are not limited to, avian inhibins, mammalian inhibins, reptile inhibins, amphibian inhibins, or fish inhibins. More specifically, mammalian inhibins include, but are not limited to, bovine inhibin, human inhibin, horseinhibin, necoinhibin, dog inhibin, rabbit inhibin, sheepinhibin, minquininhibin, foxinhibin, otterinhibin, ferret inhibin , Raccoon hibins, robin hibins, rat inhibins, mouse hibins, hamster inhibins, and porcine inhibins. Avian inhibins include, but are not limited to, ostrich inhibins, emu inhibins, rare inhibins, cucumber duck hibins, kiwi inhibins, turkey inhibins, uzurain hibins, chicken inhibins, duck inhibins, goose inhibins, and inhibins from parrots. is there.

  Preferred inhibins are birds or avian inhibins. A more preferred inhibin is chicken inhibin. Most preferably, the heterologous protein of the invention comprises an inhibin protein α subunit or fragment thereof, and a carrier protein.

  The inhibin peptide P, a fragment thereof or a substitute thereof can be isolated from animal body fluids, expressed from genetically modified cells in an expression system, or synthesized synthetically from a series of chemical reactions. it can. More specifically, inhibin fragments include, but are not limited to, the following compositions: inhibin alpha subunit; inhibin beta subunit; inhibin alpha subunit or recombinant DNA of inhibin beta subunit Fragments; synthetic peptide replicas of fragments of inhibin α subunit or inhibin β subunit; synthetic peptide replicas of N-terminal sequence of inhibin α subunit or inhibin β subunit; fragments of partially purified inhibin derived from follicular fluid; endogenous A fragment of an inhibin α subunit or inhibin β subunit; and a fragment of an exogenous inhibin α subunit or inhibin β subunit. As stated above, the inhibin fragment is most preferably a mature inhibin α subunit or a fragment thereof. It will be appreciated by those skilled in the art that inhibins include amino acid substitutions that can be made more immunogenic and more active at the receptor. It should be understood that the inhibin in a MAP composition need not be derived from the same species that administers the MAP composition. For example, the MAP composition administered to an ostrich may consist of chicken inhibin.

Carriers The MAP compositions of the present invention further comprise carriers, excipients, adjuvants, preservatives, diluents, emulsifiers, stabilizers, and other known ingredients that are known and used in prior art vaccines. Please understand that.

  The term “acceptable carrier” or “acceptable excipient” is used herein to refer to a composition suitable for use in contact with living animal or human tissue without causing adverse physiological reactions. Any, including but not limited to, water or saline, gels, ointments, solvents, diluents, liquid ointment bases, liposomes, micelles, giant micelles, etc. that do not adversely interact with other ingredients of the product Is used to mean a liquid. The MAP compositions of the present invention can be conveniently presented in unit dosage form and can be prepared by conventional formulation techniques. Such techniques include the step of mixing the active ingredient with the carrier (s) or excipient (s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers. Formulations suitable for parenteral administration include aqueous or non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents and solutes; aqueous or non-aqueous solutions that may contain suspending agents and thickening agents. There is a sterile suspension. Formulations can be provided in single-dose or multi-dose containers, such as sealed ampoules and vials, which can be freeze-dried just prior to use by adding a sterile liquid carrier, such as water for injection. It can be stored in (freeze-dried) state. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets commonly used by those skilled in the art.

  Preferred single dose formulations are those containing a single dose or unit dose, or a suitable portion thereof, of the administered ingredient. In addition to the ingredients specifically mentioned above, it should be understood that the formulations of the present invention can include other agents commonly used by those skilled in the art.

  The MAP composition of the present invention can be administered by various routes including oral, rectal, parenteral, ophthalmic, spray, nasal, intramuscular, subcutaneous, intradermal, topical, including intraoral and sublingual. it can. The MAP compositions of the present invention can be administered in a variety of forms including, but not limited to, solutions, emulsions and suspensions, fine granules, particles, microparticles, nanoparticles, and liposomes.

Adjuvant Any adjuvant system known to those skilled in the art can be administered with the composition of the present invention. Such adjuvants include, but are not limited to, Freund's incomplete adjuvant, Freund's complete adjuvant, polydisperse β- (1,4) -linked acetylated mannan (“Acemannan”), Titermax®. (CytRx Corporation polyoxyethylene-polyoxypropylene copolymer adjuvant), Chiron Corporation modified lipid adjuvant, Cambridge Biotech saponin derivative adjuvant, Bordetella pertussis dead, Gram-negative bacterial lipopolysaccharide (LPS), Large polymer anions, such as dextran sulfate, and alum, aluminum hydroxide, or aluminum phosphate And inorganic gels such as. Emulsions including but not limited to water-in-oil emulsions and water-in-oil-in-water emulsions can also be used to administer the MAP compositions of the present invention. Another adjuvant system is Freund's incomplete adjuvant. Yet another adjuvant system is Freund's complete adjuvant.

Method of Administration The MAP composition of the present invention can be administered to birds with an acceptable carrier by any means known in the art. For example, the composition can be administered subcutaneously, intraperitoneally, intraocularly, intradermally, or intramuscularly. Preferably, the composition is injected subcutaneously. The composition can be administered to the bird one or more times. Preferably, the composition is administered to the bird multiple times and boosted following the initial immunization.

  The composition can be administered to an animal whenever the animal ceases to ovulate or produce sperm due to disease or aging. The preferred age at which the composition of the invention is administered to an animal depends on the animal species used, the estrus of the animal (if any), and the purpose for which the composition is administered.

  For example, if the composition is administered to facilitate the onset of egg laying or sperm production, the composition of the invention should be administered to the bird before the bird reaches the normal age of egg laying or spring activation. is there. As mentioned above, the preferred age at which the composition of the invention is first administered to an animal is the species of animal used, the estrus of the animal (if any), the size of the bird, the inhibin peptide fragment in the composition Determined by the identity of

  As another example, when the composition is administered to promote the production of animals for the agricultural and livestock industry having a breeding season, the preferred time to administer the composition is before the start of the breeding season. In contrast, when the composition is administered to an adult animal with a decreased egg production rate or sperm production rate, the composition is administered when the decreased production is recognized as a problem.

  The preferred timing for administration to the chicken is after the bird has become immune and before it reaches reproductive maturity. The preferred minimum period between the initial injection and the booster injection is about 3 weeks. In one embodiment, the initial and booster injections are performed at 15 and 18 weeks, respectively. When a composition is administered to facilitate the onset of chicken egg production or sperm production, the preferred time to administer this composition is that for meat chickens (breeding broiler hens), 20 weeks before laying chickens. In (eg, Leghorn species), the first and one or more inoculations are performed before 18 weeks of age.

  For animals having a breeding season, the MAP composition of the present invention can be administered to birds at any age, but it is preferred to immunize the birds during the six months prior to the first breeding season of the birds. . One skilled in the art will appreciate that the average hen begins to lay eggs during its first breeding season. More preferably, the birds are immunized about 2-6 months prior to the first breeding period and then booster injections are administered at one month intervals prior to the first breeding period of the birds. Most preferably, the bird is immunized about 6 months before the first breeding period and a booster injection is administered one month before the first breeding period of the bird.

  The initial immunization includes about 0.005-1.0 mg of the MAP composition of the present invention. The booster injection will contain about 0.0025 to 1.0 mg of the MAP composition of the invention. Preferably, the initial immunization comprises about 0.01 to 0.75 mg of the MAP composition of the invention. More preferably, the initial immunization comprises about 0.02 to 0.50 mg of the MAP composition of the invention. The booster injection preferably contains about 0.005-0.5 mg of the MAP composition of the invention, more preferably about 0.01-0.025 mg of the MAP composition of the invention. In the first immunization injection, the MAP composition is emulsified in a water-in-oil (WO) or water-in-oil-in-water (WOW) emulsion, and in a booster injection, water-in-oil (WO) or water-in-oil-in-water ( It is also preferred to emulsify the MAP composition in a (WOW) emulsion. It is preferred to inject the MAP composition subcutaneously. It is preferred to inject the MAP composition subcutaneously at one location along the dorsal side of the animal, preferably the cervical region of the bird.

  The amount of the MAP composition of the invention to be administered to a bird includes the species of the bird, the age and weight of the bird, the timing of administering the composition in relation to the breeding season (if the bird has a breeding season), and It depends on the number of times the composition is to be administered. The start of the dosing schedule or treatment schedule also includes the species of the bird, the average age of the bird during the spring trigger period, the bird's family calendar (related to the family calendar of the spring trigger age), and the time when the bird hatched , Bird nutrition (sufficiently nourished birds enter the spring period before malnourished birds), bird weight, meat and fat levels, ambient lighting conditions (natural and artificial breeding The environment interacts with illuminance and the duration of the light period of the day, so it relates to these factors), the general health of the bird at the start, the immune system of the bird, the long-term health calendar of the bird, extreme It depends on the presence of weather conditions (rain, high temperature, strong winds, etc., where birds are unfamiliar, excessively rough weather over the long term), rearing conditions (overcrowding), and lack of exercise.

  One of ordinary skill in the art should be able to determine the amount of MAP composition required to elicit an immune response against the protein by a bird, using routine testing methods in view of the teachings of the present invention. .

  Another example of a method for promoting productivity is as follows. An immune effective amount of the MAP composition is administered to the mammal such that an immune response against the MAP composition occurs in the mammal. Preferably, the MAP composition consists of a mammalian inhibin peptide fragment linked to an amino acid backbone. Another preferred MAP composition consists of a peptide fragment of avian or reptile inhibin.

  For example, a brief overview of the method of the present invention that promotes the ability to produce Japanese quail, fully discussed in Example 4, is as follows. The average age of untreated quail during spring activation is approximately 6-8 weeks of age. The following is a treatment schedule for Japanese quail with an approximate weight of 0.1-0.25 pounds: the first (first) injection of about 0.025-0.1 mg of the MAP composition of the present invention at 25 days of age. Additional injections of 0.01-0.05 mg at 32-35 days of age.

  A brief overview of the method of the present invention that promotes chicken productivity, discussed in Example 2, is as follows. The average age of untreated egg-laying chickens (eg, Leghon) during the spring activation period is about 20 weeks of age. The treatment schedule for an laying chicken with an approximate weight of 2.0-3.0 pounds is as follows: At 14 weeks of age, about 0.05-0.1 mg of the MAP composition of the present invention is initially (first A) Injection, additional injection of 0.025 to about 0.05 mg at 17 weeks of age. The average age of untreated meat chickens (breeding broilers) during the spring period is approximately 23-25 weeks of age. The treatment schedule for meat chickens with approximate body weights of 3.0-4.0 pounds is as follows: At 15 weeks of age, about 0.05-0.1 mg of the MAP composition of the present invention is initially (first Injection), about 0.025 to about 0.05 mg boosted at 18 weeks of age.

  A brief summary of the method of the present invention that promotes turkey productivity discussed in Example 5 is as follows: The average age of untreated turkeys during the spring activation period is about 30 weeks of age. The treatment schedule for turkeys with an approximate weight of 9.0-12 pounds is as follows: 24 weeks of age, the first (first) injection of about 0.1 to about 0.5 mg of the MAP composition of the invention Additional injection of about 0.05 to about 0.25 mg at 27 weeks of age.

  As discussed above, the methods of the invention promoted productivity by facilitating the onset of the spring onset of animals administered the compositions of the invention. The term “promoting” with respect to the onset of spawning indicates that the spawning of the treated bird begins at least about 3% earlier than normal spawning in the untreated bird. Egg laying preferably begins at least about 5% earlier, more preferably at least 7% earlier. Egg laying begins more preferably at least about 10% earlier, and most preferably at least about 13% earlier than normal egg laying in intact birds.

  As discussed above, the method of the present invention also promoted productivity by increasing animal egg or sperm production. The term “increasing” with respect to egg production indicates that the egg production of the treated bird is increased by at least about 3% over the egg production of the untreated bird. Egg production is preferably increased by at least about 7%, more preferably by at least about 12%.

  Furthermore, as discussed above, the methods of the present invention promote productivity by facilitating the onset of animal maximum egg production. The term “promoting” with respect to the onset of maximum spawning indicates that maximum spawning of treated birds begins at least about 3% earlier than normal spawning in untreated birds. Maximum egg production preferably starts at least about 5% earlier, more preferably at least 7% earlier. Maximum egg production begins more preferably at least about 10% earlier and most preferably at least about 13% earlier than maximum egg production normally begins in untreated birds.

  Surprisingly, the compositions of the present invention are useful for increasing the lifetime egg production of birds. The term “increasing” with respect to lifetime egg production indicates that the egg production of the treated bird is increased by at least about 3% over that of the untreated bird. Lifetime egg production is preferably increased by at least about 7%, more preferably by at least about 12%. Most preferably, lifetime egg production is increased by at least about 15%.

  Surprisingly, the compositions of the present invention are useful in reducing or eliminating the need for molting hens, such as the need to extend egg continuity by providing a second cycle of egg laying. More specifically, as disclosed in the method above, when the composition described above was continuously administered to hens, the egg laying rate of the birds was not treated with the composition of the invention. Compared to the case, it remains high enough that it does not need to be molted to increase egg production. Such as hens (single-comb white leghorns, laying hens for edible eggs) when the egg-laying yield drops so that the economic cost of maintaining the birds exceeds the economic benefits obtained by the eggs produced It is common practice in the art to moult a hen. In order to “muffling” a hen, it usually begins to molt, for example, giving the bird a fasting period (eg, reducing the light part of the light-dark cycle of the day) for about 4-14 days until the wing begins to fall off. There are alternative ways of inducing molting). During the molting period, birds stop laying eggs. Egg production begins after a while after returning the birds to their normal feeding levels. The entire molting period is about two months from the start of the fasting period to the start of the next egg-laying cycle. In fact, bird egg production rates are restored. However, after chicken molting, the egg-laying rate in the next cycle is not equivalent to egg production during the first (before molting) egg-laying cycle. M.M. North and D.C. Bell, Commercial Chicken Production Manual, 4th edition, chapter 19, published by Van Norland Reinhold, New York.

  For example, chickens used to produce edible eggs begin to lay eggs in about 20 weeks and produce an economically meaningful number of eggs in about 40-50 weeks. At maximum egg production, chickens produce 8-9 eggs every 10 days. However, after about 50 weeks of egg laying, the egg production rate drops to about 60% of maximum egg production. At this point, the chicken's feeding cost is higher than the price of the egg it produces. It is usually practiced to moult chicken at this point so that the egg-laying rate increases when the chicken resumes egg-laying. Among birds, “prolonging egg laying” for chickens and quails means that egg laying is prolonged for about 1 to 4 weeks.

  Thus, the composition of the present invention reduces or eliminates the need for chickens to molte because the composition maintains egg laying rates at a higher level than if the birds were not treated with the composition. When the need for molting birds is reduced or eliminated, there is considerable savings. More specifically, during and before molting the bird, the bird is less productive than the feeding cost before molting, and then unproductive for some time after resumption of feeding. Thus, by maintaining the egg laying rate at a high level, this non-productive period of birds is shortened or eliminated, thereby reducing the cost of the producer and increasing the profit of the producer. As discussed above, since the spawning rate after molting is not equal to the spawning rate of the first spawning cycle, maintaining the spawning rate at a high level further increases the profits of the egg producer.

  Briefly, administering an effective amount of the MAP composition of the present invention to induce an immune response, followed by administering an effective amount (additional) of the MAP composition of the present invention to maintain a higher than normal egg-laying rate. This increases the bird's spawning rate, thereby avoiding the need for molting the bird.

  The method of the present invention promotes the productivity of female animals that produce inhibin, such as mammals, reptiles, and birds such as poultry. More specifically, the method promotes the production capacity of chickens, quails, ducks, geese, and turkeys. Surprisingly, the method of the present invention facilitates the onset of the animal's spring activation or first egg laying. The method of the present invention also facilitates the onset of maximum egg production in animals. Furthermore, the method of the present invention increases the number of eggs laid in an animal. Furthermore, the method of the present invention prolongs the maximum egg-laying duration of the animal. Furthermore, the method also increases the animal's lifetime egg production. In birds, the method of the present invention also improves the feed requirement of the birds. Surprisingly, the method of the present invention also reduces or eliminates the effect of such harmful egg-laying conditions on the egg-laying rate of animals exposed to harmful conditions. Such harmful conditions include, but are not limited to, high temperature, overcrowding, malnutrition, and noise.

  By immunizing an animal with a MAP composition of the invention, antibody production against the inhibin-related peptide in the MAP composition is selectively induced in the animal. Also preferably, this immunization selectively induces production of antibodies against endogenous inhibin in the animal. Although not limited to the following explanation, it is considered that the production of such an antibody by a bird shortens the period until the onset of spring or the start of egg laying. Also, although not obsessed with the following description, the production of such antibodies by an animal causes the antibody to affect or neutralize the biological activity of inhibin in the animal's bloodstream, It is thought to enhance the egg production ability or sperm production ability of animals.

  Surprisingly, the method of the present invention also improves the productivity of male animals that produce inhibin, such as mammals, reptiles, birds and the like. More specifically, the methods of the present invention increase male animal weight and blood testosterone levels. Similarly, the method of the present invention promotes the onset of male animals in the spring onset or sperm production. The method of the present invention also promotes the initiation of maximum sperm production in male animals. Furthermore, the method of the present invention increases sperm production (number of sperm) by male animals. Furthermore, the method of the present invention extends the duration of maximal sperm production in the animal. The method of the present invention also increases the ejaculation amount of male animals. Furthermore, the method improves sperm viability and properties (eg, motility) in animals. Furthermore, the method surprisingly reduces or eliminates the effects of such harmful conditions on sperm production in animals exposed to the harmful conditions. Such harmful conditions include, but are not limited to, high temperature, overcrowding, malnutrition, and noise. The method of the present invention also surprisingly increases the sexual desire of the rooster and thus increases the potential fertility of the rooster.

  The following examples help to further illustrate the invention, but at the same time are not intended to limit it in any way. On the contrary, various other embodiments that the specification may suggest to those skilled in the art after reading the description herein, without departing from the spirit of the invention and / or the appended claims, It should be clearly understood that modifications and equivalents may be used.

Generation of MAP composition shown in Formula XIV using N-terminal 26 amino acids (SEQ ID NO: 1) of mature α subunit of chicken inhibin. This antigen was generated by Midwest (St. Louis, MO). SEQ ID NO: 1 was chemically synthesized, and four such sequences were covalently linked to a lysine skeleton (Lys Lys Lys) to create the structure shown in Formula XIV. Solid phase methods known to those skilled in the art of peptide synthesis (Merrifield, RB, J. Am. Chem. Soc. 86, 1385 (1963) Stewart, JM and Young, JD “Solid”). The composition represented by Formula XIV was synthesized using “phase peptide synthesis”, a method by Pierce Chemical Co., Rockford Illinois, 1983). First, a lysine backbone (B) was synthesized, and then 26-mer amino acids (P, SEQ ID NO: 1) were added one by one on B (from the C-terminus). The entire MAP complex shown in Formula XIV was cleaved from the solid phase and then lyophilized for storage.

Administration of MAP composition shown in formula XIV to broiler hens for breeding to promote production ability Produces anti-inhibin antibody and affects production ability for breeding with MAP composition shown in formula XIV Female broilers were immunized.

Summary of Procedure 130 breeding female broilers (Cobb 500) were obtained from commercial sources. Ten hens were evenly assigned hens. The kite is a combination of about 50 square feet of slat space (covered wire mesh, Riverdale Mills) and about 50 square feet of laying (wood). At the age of 15 weeks, 40 hens (4 per pup) were randomly selected, weighed, winged, and designated as subjects.

  A drip nipple watering system (6 nipples per kite) was suspended over the vane plate (near the center of the vane) and extended across the vane in a direction perpendicular to the rear wall of the kite. Two tube-type feeders were hung on both sides of the drip nipple water feeder on the slats. Each feeder was placed approximately equidistant between the water feeder and the adjacent wall. Water was appropriately supplied by the water supply system described above.

  From the start of bird placement, the Cobb-Vantres recommended 4: 3 feeding restriction method was used. From the beginning of 13 weeks of age, the birds were weighed weekly to determine if the target weight was reached. Feeding amount was adjusted to reach the target weight. According to the arrangement in the breeding area, a standard broiler broiler mash feed (LSU BBD2) was given. On the feeding day, feeding was started around 07:00.

  Before starting egg laying by increasing the light period of the day to the light period of 16 hours: dark period to 8 hours (see below), the hens (i.e., 13-20 weeks of age) should be The period was maintained at 8 hours. During the dark period, the illuminance was less than 0.5 lux. During the light period, the auxiliary lighting supplied by the incandescent bulb was approximately 18 ± 0.5 lux (measured at night by bird head height). At 20 weeks of age, the birds were “illuminated” by giving another 5 hours of light a day. Thereafter, the light period of the day was increased by one hour per week until the light period of 16 hours: dark period of 8 hours was reached (according to Cobb-Vantless's recommendation). From the beginning of 20 weeks of age, birds were fed a standard broiler mash feed (LSU BBL) for breeding broilers.

  The normal observation and recording of birds, and the setting of the rearing environment are: AABL SOPs # AM3-0, Dairy Observations; # FR5-1, House Operations; went.

Is there an immune response in breeding broiler hens to induction by 1 of 3 doses of MAP complex ( ^1-26 INH-MAP) shown in Formula XIV or induction by Freund (control)? I tested it. According to the following treatment schedule (see protocol section below), birds selected for testing in each cage will receive the first dose as a control challenge or with one of three different doses The administration of the MAP composition represented by Formula XIV was performed. Birds remaining in each cage (n = 9, birds not treated with this MAP composition, or birds not used as controls) were not considered test animals. The first inoculation was performed at 15 weeks of age. The injection route was subcutaneous (sc) and the injection vehicle was Freund's complete adjuvant (FCA). At 18 weeks of age, a single booster injection was given at half the initial dose. Blood samples were obtained at 19, 20 and 21 weeks of age and evaluated for the presence of anti-inhibin antibodies (antibody titer). Fertility was also measured as follows. From the periodic measurement of the average pubic extension (PS) of each hen, and from the calculation of the average age of first egg laying (FIRST) and the average age of egg laying rate 25% (T-FIVE) Estimated. Average weekly and cumulative Hendy egg production (HDEP) was also determined over 19 weeks of egg production. Weight gain (BWG, expressed as the difference in body weight at the time of first dose induction and at the end of the study) and mortality (MART) were also examined. Each treatment group consisted of 10 hens. Therefore, including the Freund control group, 40 birds were divided into 4 treatment groups as follows.

  The first inoculation was performed at 15 weeks of age. The injection route was subcutaneous (sc) and the injection vehicle was Freund's complete adjuvant (FCA). At 18 weeks of age, booster injections were given at half the initial dose. Additional injections were also subcutaneous (sc) and the injection vehicle was Freund's incomplete adjuvant (FIA). The control group of birds received FCA as the first inoculation (sc) at 15 weeks of age and FIA as a booster (sc) at 18 weeks of age. The three groups treated with different doses of the MAP composition (Formula XIV) and the control treatment group were identified by wingband numbers rather than showing doses to keep the field workers unaware. did. With AABL SOP # FR2-0, Random Alert of Leg / Wing Badge Codes to Treatment Groups, the study administrator assigned doses to the wing cords. All treatments (including controls) were performed on behalf of one bird per treatment group per treatment among all the cages, for a total of 10 birds per treatment. Birds remaining in each cage (n = 9, birds not treated with MAP composition (Formula XIV), or birds not used as controls) were not considered test animals. Treatment groups were assigned as described in AABL SOP # FR3-0, Allotment of Treatments to Broiler Breeders with Penns. When the treatment was applied, all hens were weighed according to AABL SOP # AM4-0, Animal Weighing-Broiler Breeders. Each treatment was administered as described in AABL SOP # FR4-0, Broiler Breeder Subcutaneous Injection Protocol.

  Blood samples were obtained from each test hen at 19, 20 and 21 weeks of age (1, 2 and 3 weeks after the booster inoculation). Blood collection was performed as described in AABL SOP # FR6-0, Blood Collection, and blood was processed by AABL SOP # LE4-0, Blood Processing. At the end of the test, the hens were all weighed as described in AABL SOP # AM4-0, Animal Weighing-Broiler Breeders.

  A total of 8 PS measurements were made on all test birds at about 23-28 weeks of age. The first 6 PS palpations were performed approximately every 4 days, and the last 2 were performed at approximately 7 day intervals. This measured value was averaged for one bird, and the PS average value for each bird was used as an observation for statistical analysis. Daily mortality records were maintained and all birds that died during the study were examined for abnormalities, lesions, and possible causes of death (see below).

  The studied treatment is on the main plot and the time of blood collection (repeated measurements, 19, 20 and 21 weeks of age, ie 1, 2 and 3 weeks after the booster) and the interaction of the studied time and treatment on the split plot The difference in antibody titer between injection treatments (0, control and 3 MAP (formula XIV) doses) was evaluated using ANOVA incorporating the split method. Differences in PS, FIRST, T-FIVE, cumulative HDEP, BWG and MORT between treatments were evaluated using ANOVA incorporating a fully randomized approach that considered the main effects of injection treatment. In order to divide the differences in these variables when applicable, Duncan's new multi-range test was used to post-test the treatment group means.

  Measurements of the onset of the spring phase including PS, FIRST, and T-FIVE were obtained. Egg production was assessed by calculating weekly and cumulative HDEP from daily egg laying observations during 19 weeks of egg laying. Body weight gain (BWG, expressed as the difference in body weight at the time of first dose induction and at the end of the study) and MORT were also examined. A comparison was made between the three doses of antigen and the control.

Anti-inhibin antibody titer (antibody titer)
Examination of the effect of injection treatment on antibody titers with ANOVA showed significant differences (P <0.0003) between treatment groups. Divide this difference by Duncan's test after ANOVA (see below), when compared to the mean TITER response of the control, the ^1-26 INH-MAP dose was the lowest bird (initially 0 per bird) .05 mg) and the highest bird (0.20 mg per bird for the first time) showed a high average TITER response (P <0.01). Birds inoculated with an intermediate ^1-26 INH-MAP dose (initially 0.10 mg per bird) also showed an average TITER response that was robust (almost 30 times higher than the control). Despite being statistically similar to the high TITER found in birds receiving two other antigen doses, this could not be statistically divided from the controls. Because of the small sample size, this statistical oddity is likely to be explained. As expected, TITER decreased with time (P <0.02) after induction of additional doses, primarily due to the effects of antigen treatment (data not shown). There was no significant interaction with mean TITER between the injection treatment and the time after boost.

Measurement of the spring onset period Periodic measurement of the average pubic dilatation (PS) of each hen, and calculation of the average age of first egg laying (FIRST) and the average age of the egg laying rate of 25% (T-FIVE) The start of the triggering phase was estimated.

Pubic extension (PS)
The pubic interval, pubic extension or PS is a standard item measured in the poultry industry to assess the status of sexual development in laying hens. Typically, PS increases from the width of one finger at 17 weeks of age (about 2 cm) to the width of three fingers at the time of egg laying (23 to 25 weeks of age) (about 5 cm), Ross Breeder Management Guide, 2000, p. 40. A total of 8 PS measurements were made on all test birds at about 23-28 weeks of age. The first 6 times were performed at a frequency of about every 4 days, and the last 2 times were performed at an interval of about 7 days. The first 4 PS measurements were taken during the 2 weeks prior to the start of egg laying, and the last 4 were taken during the subsequent 2.5 weeks. Thus, PS measurements are basically from the time of first egg laying by any test hen (low dose ^1-26 INH-MAP bird, 25 weeks 3 days old) to the first 2.5 weeks of egg laying. Performed during the bird population age range. PS measurement values were averaged for one bird, and the PS average value for each bird was used as an observation for statistical analysis. When compared to the control PS response, the average PS was similarly increased in all birds receiving three doses of ^1-26 INH-MAP (Formula XIV) (range: 17-38%, P < 0.01).

Since the increase in PS is considered to indicate the hen's readiness to begin spawning, the PS measured here (average in each bird over 8 palpations) is the first 4 weeks of the spawning season There was a correlation with cumulative HDEP. PS is the correlation in each injection treatment group (r ² = 0.91, P <0.01 for the correlation in the control group alone, and the lowest, medium and highest doses of ^1-26 INH-MAP In the groups, r ² = 0.91, P <0.01, r ² = 0.65, P <0.08, r ² = 0.85, P <0.002), and all test birds, respectively. (R ² = 0.83, P <0.0001) was consistently high with early egg-laying.

FIRST and T-FIVE
When compared to the control response, FIRST and T-FIVE measurements decreased in the birds that received each of the three doses of ^1-26 INH-MAP, and the mean in the intermediate dose group The FIRST response was statistically different from the control response at a level of P <0.10 (FIG. 1). Due to the small scale of sampling, it was clearly impossible to find any further statistical differences in these measurements during the spring period. Nevertheless, these measurements, which show that the average age at the start of the spring exercise period, has been promoted are the best that we have done in any test. Furthermore, this FIRST and T-FIVE finding fully supports the PS finding that demonstrates a dramatic increase in mean pubic dilatation in ^1-226 INH-MAP treated birds (see discussion above) )

Spawning measurement
Weekly HDEP
During the 8th week of egg laying, the two high dose ^1-26 INH-MAP groups showed higher mean PEP (P <0.10) than that seen in the control group. And the lowest dose ^1-26 INH-MAP treated group showed an intermediate HDEP response, which was about 20% higher than the control response, but the control group or two other ^1-26 INH- There was no statistical difference from the MAP treatment group. However, a significant and statistically significant increase in the weekly HDEP (P <0.05-) in the week 9 of spawning and thereafter (until the end of the study) in one or more ^1-26 INH-MAP administered groups 0.01) was evident in 8 of the 11 weeks examined. Specifically, the egg-laying weeks were weeks 9, 10, 12, 13, 14, 15, 16, and 19.

Cumulative HDEP
Analysis of mean cumulative HDEP (at 19 weeks of egg laying) dramatically increased overall egg production in all three ^1-26 INH-MAP groups when compared to control responses (P <0.05). ) (See below). The cumulative HDEP increase over the control of the birds treated with the lowest, middle and highest doses of ^1-26 INH-MAP was about 49%, about 62% and about 64%, respectively.

Measuring weight gain and mortality
BWG
There was no difference between the control group and the ^1-26 INH-MAP group in weight gain from the time of the first dose induction (15 weeks of age) to the end of the test (19 weeks of laying) (data not shown). This suggested that treatment with the inhibin-based vaccine did not change the salvage value of hens measured at the mid-point (19 weeks) of the complete egg-laying cycle (40 weeks).

MORT
Mortality was also unaffected by vaccine treatment (data not shown). These findings support the claim that aggressive immunization against inhibin does not affect viability.

Administration of the MAP complex shown in Formula XIV to breeding broiler hens to promote productivity . A second major clinical trial similar to the trial described in Example 2 was conducted in chickens.

Animals and Management 400 13-week-old Cobb-500 hens (350 test animals and 50 surpluses, which are within ± 10% of the recommended target body weight at 13 weeks of age, selected at a commercial farm. (See below) was randomly divided into 16 pieces. At 15 weeks of age, 350 hens (14 cages) were designated as subjects and attached with a wing badge with a different color and number to provide a permanent identification of each bird. Assigned. The remaining 50 birds initially raised at 13 weeks of age (2 wings of 25 each) were maintained in the same manner as the test hens. A few surplus birds instead of test birds that died between 13-15 weeks of age (before treatment application) or test birds that were decimated at the time of the first injection (15 weeks of age, see below) It was used. There was no further exchange of test animals after the first inoculation.

  The dredge and water supply system was as described in Example 2. A total of 18 individual trap nests were placed in a two-row system along the rear wall of each cage. Water was supplied as appropriate throughout the test. From the start of bird placement (13 weeks of age), the daily (ED) feeding restriction method, Cobb-Vantres recommended feeding restriction method, was used. From 13 weeks of age to the start of egg laying (23 weeks and 5 days of age), the birds were weighed weekly to determine if the target weight was reached. Feeding amount was adjusted to reach the target weight recommended by the trader.

  Commercially formulated and mixed breeder broiler feed (GMP certified, NAMCO, OH) was provided throughout the experiment. From the start of laying hens at 13 weeks of age to Wednesday after the Henday spawning rate by the bird population reached 5%, feed for the growing season / growing season was given, and the feed was used for the growing season / growing season on that Wednesday. I changed it for the breeding season. Daily feeding started about 30 minutes after lighting (see below).

  When selected in a commercial facility (at 13 weeks of age), the hens were raised under dark-out conditions of 8 hours of light during the day, so this condition (light hours of 8 hours) : A period of 16 hours in the dark period) until 20 days and 5 days of age. During the dark period, the illuminance was less than 0.5 lux. During the light period, the auxiliary lighting supplied by the incandescent bulb was about 15 ± 0.5 lux (measured at night by bird head height). At 20 weeks and 5 days of age, the light period of the day was increased by 5 hours (the light period of the day was changed from 8 hours to 13 hours, 60 lux). Then, at 21 weeks and 5 days of age, the day's light duration is increased by 1 hour, then 30 minutes every week until a period of 16 hours of light: 8 hours of darkness is reached (ie 25 weeks and 5 days of age) It gradually increased the light period.

  Normal observation and recording of birds and setting of conditions for breeding were performed as described in Example 2. During the study, it was determined that unexpected events, including bird damage, disease, etc., may have to be excluded from the study. Therefore, exclusion from all bird tests followed the procedure described in Example 2. In addition, a veterinary autopsy report with all possible bird deaths describing possible causes of death was obtained.

Injection treatment dose and dosing schedule The test antigen used was the first 26 amino acids of the chicken inhibin alpha subunit in the form of a 4-mer multi-antigen peptide (MAP) ( ^1-26 INH-MAP, Formula XIV). It was. In addition to the vehicle control (see below), the following two test article treatment doses were administered.

Each bird to be inoculated (initial and additional) was injected with an injection volume of 1.0 mL (appropriate concentration of test article in 0.5 mL of aqueous solution + 0.5 mL of appropriate adjuvant, see below). . ^{A 1-26} INH-MAP aqueous solution was prepared using 5% dextrose. The test vehicle and the control inoculum for the first injection were Freund's complete adjuvant (FCA). The first inoculation (subcutaneous (sc)) was performed at 15 weeks of age. At 18 weeks of age, a booster injection (half the concentration used to induce the first dose with the test article, see above) was given. The additional injection route was also subcutaneous (sc) and the injection vehicle was Freund's incomplete adjuvant (FIA). As controls, initial subcutaneous injection of FCA 0.5 mL + distilled water 0.5 mL was given at 15 weeks of age, and additional subcutaneous injection of FIA 0.5 mL + distilled water 0.5 mL was given at 18 weeks of age.

  At the first treatment application (first injection at 15 weeks of age), the population of birds is thinned to 350 hens and distributed equally to 14 pups (25 hens per pup) according to established protocols. )did. The injection treatment groups were identified by the color and number of the wing badge, rather than showing and identifying the dose, in order to keep the field workers unaware. All injection treatments were applied to appropriate birds in all cages. In other words, there were 5 birds per treatment group and 70 birds per treatment as a whole. The protocol was as described in Example 2.

Blood samples for anti-inhibin antibody determination Blood samples were obtained from each hen 18 weeks 4 days and 20 weeks old (ie, 4 days and 2 weeks after the booster inoculation). Blood collection was performed as described in Example 2.

To minimize nest acclimatization and hen spawning behavior on the spawning bed (in the scratch area or on the wire mesh slats), trap nests should be applied prior to spawning. Used to acclimate hens. Acclimatization was performed daily from 21 weeks 1 day to 21 weeks 4 days old and also from 22 weeks 1 day to 22 weeks 4 days old. Therefore, acclimatization to the hen's nest was terminated approximately one week before the first egg laying was observed.

  The test spawning period began after the first egg laying by any bird was observed. This occurred when the bird population was 23 weeks and 5 days old. The test was terminated after 16 weeks of the spawning period (at about 40 weeks of age).

Measured variable
Spring period measurement:
To determine the state of sexual development of pubic dilated females, the pelvic ("pin") bones interval of each 17-26 week old hen was measured according to the procedure described in Example 2. Pubic dilation (PS) was measured in units of half a finger ranging from one finger (no sexual development) to five fingers (maximum sexual development).

FIRST and TFIVE
The average age (days) of the first egg laying (FIRST) and the average age (days) at which the individual reached the egg-laying rate of 25% (TFIVE) were calculated for the hens in each treatment group.

Periodic egg collection (minimum 4 times a day) was initiated after the first egg collection from any bird was observed at the Hendi spawning trap nest. From the start to the end of the test (16 weeks of spawning season), the number of eggs laid daily was recorded. Daily egg data was used to calculate weekly as well as cumulative Henday egg production (HDEP).

Egg weight (EWT)
To assess the effect of vaccine treatment on initial egg size, the weight of the first 12 eggs produced by each hen in each injection treatment group (at the trap nest) was measured individually. If eggs from individual hens were collected and the average weight of the last two eggs did not reach or exceed 47.3g (the “standard” egg weight as an industry standard), the 12th The weight was measured beyond the egg. Once a given hen produced the first egg, egg weight was measured for up to 4 weeks. To assess the effect of the vaccine treatment on the size of mature hen eggs, the daily egg weight (g) of each hen during the four-day egg-laying period from the beginning of 32 weeks of age was also recorded. Marks indicating the color and wing badge number of the hen treatment group and the hen's wing number were placed on the eggshell surface. This allowed a given hen's egg to be associated with that hen's specific injection procedure by referring to the blinded assignment of the wing badge.

Body weight Body weight (BWT) was measured weekly from 13 to 26 weeks of age according to the procedure of Example 2.

Mortality and necropsy Daily mortality MORT records were continued to examine all birds that died during the study for abnormalities, lesions, and possible causes of death. Death was recorded as KT (sacrifice by life span, no autopsy required), KM (slaughter by disease, necropsy required), D (death, necropsy required) or RM (mechanically excluded, no autopsy required).

Anti-inhibin antibody titers Relative antibody titers to inhibin α subunit were measured by ELISA (Viro Dynamics) in blood samples collected at 18 weeks, 4 days and 20 weeks of age. The collection of blood samples corresponded to 4 days and 2 weeks after the additional injection, respectively.

Statistical analysis Satterlee et al. (2002) Politry Sci. 81: 519-528 were analyzed for titer data (PS, FIRST, and TFIVE), HDEP (weekly and cumulative), EWT, MORT, and ELISA antibody titer data by a procedure similar to that described in 81: 519-528. .

Results and Discussion With a low dose of ^1-26 INH-MAP (0.05 mg), the onset of PS was significantly earlier than 21 weeks old (mostly P <0.05) and persisted until 24 weeks of age. . High doses ( ^1-26 INH-MAP 0.1 mg) increased PS at this time, but did not produce a statistically significant difference from this increase.

  This population of birds is light stimulated at 5 weeks of age 20 weeks, so the first statistical in PS between control and inhibin immunized hens only 2 days later (at 21 weeks of age) From the finding that the difference has occurred, vaccination against inhibin has increased the effect of lighting and the increase in FSH required to mature the ovary (by FSH induction, follicular recruitment and accelerated development). It suggests that it may promote the spring puberty period, to some extent regardless of its known effects.

When compared to the control response, there was a significant increase in post-peak HDEP egg laying rate for the particular week and for the particular dose in the ^1-26 INH-MAP immunized hen group. Specifically, all of this difference occurred during the second half of the study (ie, between the 9th and 16th weeks of the spawning season), which indicated that ^1-26 INH-MAP extended the duration of spawning. Is suggested. As with the control in this study, the HDEP peak of the breeding Cobb broiler is known to occur around the 8th week of the spawning season. The difference in HDEP after peak seen between hens treated with low doses of ^1-26 INH-MAP and controls was shown in week 9 (P <0.10), week 13 (P <0) .05), a dramatic increase in HDEP (about 10% in each case) seen at week 16 (P <0.05).

In support of the previously presented weekly HDEP data, when compared to the control response, both doses of ^1-26 INH-MAP reduced the number of hens that did not lay eggs in the last two weeks of the study. (3.7 and 2 times at low and high doses, respectively). The low dose ^1-26 INH-MAP response was a statistically significant reduction (P <0.03) when compared to the control. These data demonstrate a significant reduction in the number of hens vaccinated against inhibin but not laying eggs, which is a direct economic benefit to producers.

  In addition, for hens that have reached or exceeded the target weight (BWT), as opposed to heavier hens, the immunization against inhibin is for breeding in hens whose weight is below the target weight upon lighting. The effect of broilers on egg laying was also examined. Specifically, using the data for all 16 weeks of egg laying, and data for only the last 8 weeks of laying (9th to 16th weeks of laying), in the “light” vs. “heavy” hen subpopulation, The effect of the vaccine on (hen-housed) egg-laying number (HHEP) was tested, a sufficient number of observations for simplicity and to enable meaningful statistical tests in treatment groups by BWT classification To assure (hens), data from low and high dose hens for each test article were combined.

  The target weight of a breeding Cobb-500 hen at 21 weeks of age (when illuminated) is 2320 g. The average BWT of the bird population at this time was 2132 g, that is, about 92% of the target weight. Therefore, within each injection treatment group, hens with BWT ≧ 2132 g at 21 weeks of age were considered “heavy” hens and the remaining hens with BWT <2132 g were considered “light” hens. Using this classification system, the average BWT for the light and heavy hen treatment groups was 1947 g and 2305 g, respectively (ie, about 84% and about 99% of the target BWT, respectively).

Light BWT hens treated with ^1-26 INH-MAP were statistically superior to light BWT controls (generally P <0.05) (i.e., the entire study, all eggs laid for 16 weeks). data (P <0.08), or the second half of the laying period only, when considering the data of laying the 9-16 week (P ^<0.05), higher in HHEP of 1-26 INH-MAP immunized birds ) This increase in HHEP over the control response was significant (about 14%) in each of the two test periods. In fact, immunization of light hens against inhibin resulted in high HHEP, which was remarkably similar to the high HHEP observed in heavy hens regardless of their injection treatment. In contrast, no difference in HHEP was detected between the heavy control group and either of the two heavy hen groups immunized with inhibin (all 16 weeks laying or weeks 9-16 laying). These findings suggest that producers can gain significant economic benefits by deliberately feeding birds to lighter BWT upon light stimulation.

  For chicken inhibin α subunit in blood samples collected at 18 weeks of 4 days (4 days after boost) and 20 weeks of age (2 weeks after boost) as measured by a commercially developed ELISA Relative mean antibody titers revealed that inhibin-based antigens (regardless of dose) showed strongly stable antibody titer ELISA responses (P <0.01) when compared to control responses.

To assess the effect of vaccine treatment on initial egg size, the weight of the first 12 eggs produced by each hen in each injection treatment group (at the trap nest) was measured individually. Vaccine treatment does not affect the EWT of the first 12 eggs (data not shown) and the hen's egg age reaches 47.3 g, the industry standard “configurable” egg weight Was not affected (data not shown). This finding suggests that there is no negative effect of vaccination on egg size, a variable that can greatly affect egg hatchability. Also, the injection treatment did not change the size of the eggs at maturity (peak egg production, 32 weeks of age), which was shown by no difference in EWT between treatment groups (data is Not shown). Weekly BWT (15-26 weeks of age), and MORT for the study (from first dose induction to end of study, 16 weeks laying) were also unaffected by ^1-26 INH-MAP vaccine treatment (data not shown) ).

Promotion of quail production ability Using the ^1-26 INH-MAP composition of the present invention as an antigen, immunization of female Japanese quail prior to the spring activation period against the level of circulating inhibin and thereby initiating egg production in quail treated Facilitate. The method described in Example 2 is followed with the following exceptions. The average age of untreated quail in the spring activation period is about 6-8 weeks of age. The treatment schedule for Japanese quail with an approximate weight of 0.1-0.25 pounds is as follows. The first (first) injection of about 0.025-0.1 mg of the present invention ^1-26 INH-MAP composition at 25 days of age, and a booster injection of 0.01-0.05 mg at 32-35 days of age.

Promoting turkey productivity Using the ^1-26 INH-MAP composition of the present invention as an antigen, immunization of female turkeys prior to the spring activation period against circulating inhibin levels, thereby initiating spawning of turkeys treated Facilitate. The method described in Example 2 is followed with the following exceptions. The average age of untreated turkeys during the spring exercise period is approximately 30 weeks of age. The treatment schedule for turkeys weighing approximately 9.0-12 pounds is as follows. The first (first) injection of about 0.1 to about 0.5 mg of the ^1-26 INH-MAP composition of the invention at 24 weeks of age, and a booster injection of about 0.05 to about 0.25 mg at 27 weeks of age.

  All cited patents, publications and abstracts are incorporated herein by reference in their entirety. It will be appreciated that the foregoing description relates only to preferred embodiments of the invention and that numerous changes or modifications can be made thereto without departing from the spirit and scope of the invention as set forth in the appended claims. I want you to understand.

FIG. 1 shows the average age of first egg laying (FIRST) (open profile only) in birds injected with ^1-26 INH-MAP at doses of 0.05, 0.1 or 0.2 mg per bird. ) Histogram bar) and the average age (T-FIVE) (shaded histogram bar) with an egg-laying rate of 25%. Statistical differences are observed in bars with different letters (a, b) (P <0.10).

Claims (20)

  A composition comprising a multi-antigen peptide comprising at least two inhibin-related peptides linked to a multiple linker backbone. The composition of claim 1 comprising a multi-antigen peptide represented by Formula I.

[Wherein B is a multiple linker backbone, n is an integer from 2 to about 20, each L is a covalent bond or linking group, which may or may not be present, Is an inhibin related peptide having from about 4 to about 115 amino acid residues or a conservative substitution thereof] The composition of claim 2, wherein B is 3, L is absent, n is 4, and the composition is represented by formula VII.

  4. The composition of claim 3, wherein at least one P is SEQ ID NO: 1 or a conservative substitution thereof. The composition according to claim 1, wherein the composition is represented by any one of the following formulae.

[Wherein m is an integer from 0 to about 20];

[Wherein n is an integer from 1 to 20 and a is 1 or 2];

[Wherein P is an inhibin related peptide having from about 4 to about 115 amino acid residues, or a conservative substitution thereof, B is a multiple linker backbone, n is an integer from 1 to 20, each L Is a covalent bond or a linking group, which may or may not be present, y is 1 or 2, and x is an integer of 1 to 3] 5. A composition according to claim 4 represented by formula XIV.

The composition of claim 4 represented by formula XV.

  Administering to the animal an effective amount of a composition comprising a multi-antigen peptide comprising at least two inhibin-related peptides linked to a backbone and an acceptable carrier, said amount promoting productivity in said animal A method of promoting productivity that is effective to do. 9. The method of claim 8, wherein the composition comprises a multiple antigen peptide represented by Formula I.

[Wherein B is a multiple linker backbone, n is an integer from 2 to about 20, each L is a covalent bond or linking group, which may or may not be present, Is an inhibin related peptide having from about 4 to about 115 amino acid residues or a conservative substitution thereof] 10. The method of claim 9, wherein B is 3, L is absent, n is 4, and the composition is represented by formula VII.

The method according to claim 8, wherein the composition is represented by any one of the following formulae.

[Wherein m is an integer from 0 to about 20];

[Wherein n is an integer from 1 to 20 and a is 1 or 2];

[Wherein P is an inhibin related peptide having from about 4 to about 115 amino acid residues, or a conservative substitution thereof, B is a multiple linker backbone, n is an integer from 1 to 20, each L Is a covalent bond or a linking group, which may or may not be present, y is 1 or 2, and x is an integer of 1 to 3] The method of claim 10, wherein the composition is represented by Formula XIV.

The method of claim 10, wherein the composition is represented by Formula XV.

  14. A method according to any one of claims 8 to 13, wherein the promotion of production capacity comprises an increase in animal egg production.   The method of claim 8, wherein the animal is a bird.   16. The method of claim 15, wherein the bird is a chicken, turkey, quail, goose or duck.   Use of a composition according to any one of claims 1 to 7 in the preparation of a vaccine useful for promoting animal production.   18. Use according to claim 17, wherein the promotion of production capacity comprises an increase in animal egg production.   18. Use according to claim 17, wherein the animal is a bird.   20. Use according to claim 19, wherein the bird is a chicken, turkey, quail, goose or duck.

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